Pyridyl Reverse Sulfonamides For HBV Treatment

ABSTRACT

The present invention includes a method of inhibiting, suppressing or preventing HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of at least one compound of the invention.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/097,854, filed Dec. 30, 2014. The contents of this applicationare herein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Chronic hepatitis B virus (HBV) infection is a significant global healthproblem, affecting over 5% of the world population (over 350 millionpeople worldwide and 1.25 million individuals in the U.S.).

Despite the availability of a prophylactic HBV vaccine, the burden ofchronic HBV infection continues to be a significant unmet worldwidemedical problem, due to suboptimal treatment options and sustained ratesof new infections in most parts of the developing world. Currenttreatments do not provide a cure and are limited to only two classes ofagents (interferon and nucleoside analogues/inhibitors of the viralpolymerase); drug resistance, low efficacy, and tolerability issueslimit their impact. The low cure rates of HBV are attributed at least inpart to the presence and persistence of covalently closed circular DNA(cccDNA) in the nucleus of infected hepatocytes. However, persistentsuppression of HBV DNA slows liver disease progression and helps toprevent hepatocellular carcinoma. Current therapy goals for HBV-infectedpatients are directed to reducing serum HBV DNA to low or undetectablelevels, and to ultimately reducing or preventing the development ofcirrhosis and hepatocellular carcinoma.

There is a need in the art for novel therapeutic agents that treat,ameliorate or prevent HBV infection. Administration of these therapeuticagents to an HBV infected patient, either as monotherapy or incombination with other HBV treatments or ancillary treatments, will leadto significantly improved prognosis, diminished progression of thedisease, or enhanced seroconversion rates.

SUMMARY OF THE INVENTION

Provided herein are compounds useful for the treatment of HBV infectionin a subject in need thereof.

In one aspect, provided herein are compounds of Formula I:

or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein are compounds of Formula II:

or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein are compounds of Formula III:

or a pharmaceutically acceptable salt thereof.

Also provided herein are compositions comprising a compound providedherein (also referred to herein as “a compound of the invention”), or asalt, solvate, or N-oxide thereof. In one embodiment, the composition ispharmaceutical and further comprises at least one pharmaceuticallyacceptable carrier.

In another aspect, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of a compound of theinvention.

In still another aspect, provided herein is a method of reducing theviral load associated with an HBV infection in an individual in needthereof, comprising administering to the individual a therapeuticallyeffective amount of a compound of the invention.

In yet another aspect, provided herein is a method of reducingreoccurrence of an HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound of the invention.

Also provided herein are methods of reducing an adverse physiologicalimpact of an HBV infection in an individual in need thereof, comprisingadministering to the individual a therapeutically effective amount of acompound of the invention.

In another aspect, provided herein is a method of inducing remission ofhepatic injury from an HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound of the invention.

In yet another aspect, provided herein is a method of reducing thephysiological impact of long-term antiviral therapy for HBV infection inan individual in need thereof, comprising administering to theindividual a therapeutically effective amount of a compound of theinvention.

In still another aspect, provided herein is a method of prophylacticallytreating an HBV infection in an individual in need thereof, wherein theindividual is afflicted with a latent HBV infection, comprisingadministering to the individual a therapeutically effective amount of acompound of the invention.

Any of the above methods may further comprise administration to theindividual at least one additional therapeutic agent. In an embodiment,the additional therapeutic agent may be selected from, but not limitedto, the group consisting of a HBV polymerase inhibitor, immunomodulatoryagents, pegylated interferon, viral entry inhibitor, viral maturationinhibitor, capsid assembly modulator, reverse transcriptase inhibitor, acyclophilin/TNF inhibitor, a TLR-agonist, an HBV vaccine, and agents ofdistinct or unknown mechanism, and a combination thereof.

In another embodiment, the at least one additional therapeutic agent isselected from the group consisting of an HBV vaccine, HBV polymeraseinhibitor, interferon, pegylated interferon, viral entry inhibitor,viral maturation inhibitor, capsid assembly modulator, reversetranscriptase inhibitor, a TLR-agonist, and agents of distinct orunknown mechanism, and a combination thereof.

In still another embodiment, the additional therapeutic agent isselected from the group consisting of a HBV polymerase inhibitor,interferon, viral entry inhibitor, viral maturation inhibitor, capsidassembly modulator, reverse transcriptase inhibitor, a TLR-agonist, andagents of distinct or unknown mechanism, and a combination thereof.

In another embodiment, the pegylated interferon is pegylated interferonalpha (IFN-α), pegylated interferon lambda (IFN-λ), or pegylatedinterferon gamma (IFN-γ).

In yet another embodiment, the reverse transcriptase inhibitor is atleast one of Zidovudine, Didanosine, Zalcitabine, ddA(2′,3′-dideoxyadenosine), Stavudine, Lamivudine, Abacavir,Emtricitabine, Entecavir, Apricitabine, Atevirapine, ribavirin,acyclovir, famciclovir, valacyclovir, ganciclovir, valganciclovir,Tenofovir, Adefovir, cidofovir, Efavirenz, Nevirapine, Delavirdine, orEtravirine.

In still another embodiment, the compound and the at least oneadditional therapeutic agent are co-formulated.

In yet another embodiment, the compound and the at least one additionaltherapeutic agent are co-administered.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a general scheme used to prepare selected compounds of theinvention.

FIG. 2 shows a second general scheme used to prepare selected compoundsof the invention.

FIG. 3 shows a third general scheme used to prepare selected compoundsof the invention.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compounds that are useful in the treatment andprevention of HBV infection in man. In a non-limiting aspect, thesecompounds modulate or disrupt HBV viral replication to afford defectivevirion with greatly reduced virulence. The compounds of the inventionhave potent antiviral activity, exhibit favorable metabolic, tissuedistribution, safety and pharmaceutical profiles, and are suitable foruse in man.

HBV, a causative agent of acute/chronic hepatitis, consists of apartially double-stranded 3.2 kb circular DNA from which four proteinsare synthesized: the core, polymerase, surface antigen, and X-geneproduct.

Four promoters with unique functions have been identified in the HBVgenome. The pregenomic/core promoter directs the synthesis of 3.6 kbmRNA which contains all the genetic information encoded by the virus.This RNA serves as a replication intermediate and as a template for thesynthesis of core and polymerase. The S promoter and the pre-S promoterdirect the synthesis of 2.1 and 2.4 kb RNAs utilized for the generationof pre-S1, pre-S2, and S proteins. X promoter directs the transcriptionof 0.9 kb RNA specific for the synthesis of X gene product.Liver-specific and differentiation state-specific utilization of thesepromoters are regulated by the two enhancer elements, i.e., enhancer I(ENI) and enhancer II (ENII). These enhancers along with HNF-1(hepatocyte nuclear factor-1) binding element are largely responsiblefor the restricted tropism of HBV to hepatocytes.

The mechanism of HBV replication differs from that of other DNA virusesin that, like retroviruses, the reverse transcription step is involved.Upon infection of the hepatocytes, a partially double-stranded genome isconverted to a complete double-stranded circular, supercoiled DNA.Employing this as a template, 3.6 kb RNA, which is called the pregenome,is transcribed. The pregenome is packaged into a nucleocapsid and isreverse-transcribed using polymerase as an initiation primer to generatethe minus-strand, single-stranded DNA. The polymerization of the secondstrand follows until approximately half of the genome is synthesized,resulting in the generation of partially double-stranded circulargenome, which is coated and secreted by the infected cells.

In one aspect, the compounds of the invention are useful in HBVtreatment by disrupting, accelerating, reducing, delaying or inhibitingnormal viral replication, thereby inducing aberrant viral replicationand leading to antiviral effects such as disruption of virion assemblyor disassembly, virion maturation, or virus egress.

In one embodiment, the compounds of the invention disrupt viralreplication when the virion is immature. In another embodiment, thecompounds of the invention disrupt viral replication when the virion ismature. In yet another embodiment, the compounds of the inventiondisrupt viral replication during viral infectivity. In yet anotherembodiment, the disruption of viral replication attenuates HBV viralinfectivity or reduces viral load. In yet another embodiment,disruption, inhibition, delay or reduction of viral replicationeradicates the virus from the host organism. In yet another embodiment,eradication of the HBV from a host advantageously obviates the need forchronic long-term therapy or reduces the duration of long-term therapy.

In one embodiment, the compounds described herein are suitable formonotherapy and are effective against natural or native HBV strains andagainst HBV strains resistant to currently known drugs. In anotherembodiment, the compounds described herein are suitable for use incombination therapy.

In another embodiment, the compounds of the invention can be used inmethods of modulating (e.g., inhibit, disrupt or accelerate) theactivity, stability, function, and viral replication properties of HBVcccDNA. In yet another embodiment, the compounds of the invention can beused in methods of diminishing or preventing the formation of HBVcccDNA.

In another embodiment, the compounds of the invention can be used inmethods of modulating (e.g., inhibit, disrupt or accelerate) theactivity of HBV cccDNA. In yet another embodiment, the compounds of theinvention can be used in methods of diminishing or preventing theformation of HBV cccDNA.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and peptide chemistryare those well-known and commonly employed in the art.

As used herein, the articles “a” and “an” refer to one or to more thanone (i.e. to at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.Furthermore, use of the term “including” as well as other forms, such as“include”, “includes,” and “included,” is not limiting.

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent on the context inwhich it is used. As used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, the term “about”is meant to encompass variations of ±20% or ±10%, more preferably ±5%,even more preferably ±1%, and still more preferably ±0.1% from thespecified value, as such variations are appropriate to perform thedisclosed methods.

As used herein, the term “capsid assembly modulator” refers to acompound that disrupts and/or accelerates and/or inhibits and/or hindersand/or delays and or reduces and/or modifies normal capsid assembly(e.g., during maturation) and/or normal capsid disassembly (e.g., duringinfectivity) and/or perturbs capsid stability, thereby inducing aberrantcapsid morphology and function. In one embodiment, a capsid assemblymodulator accelerates capsid assembly and/or disassembly, therebyinducing aberrant capsid morphology. In another embodiment, a capsidassembly modulator interacts (e.g. binds at an active site, binds at anallosteric site, modifies and/or hinders folding and the like) with themajor capsid assembly protein (CA), thereby disrupting capsid assemblyand/or disassembly. In yet another embodiment, a capsid assemblymodulator causes a perturbation in structure and/or function of CA(e.g., ability of CA to assemble, disassemble, bind to a substrate, foldinto a suitable conformation, or the like), which attenuates viralinfectivity and/or is lethal to the virus.

As used herein, the term “literature-described capsid assemblymodulator” refers a capsid assembly modulator that is not a compound ofthe present invention.

As used herein, the term “treatment” or “treating,” is defined as theapplication or administration of a therapeutic agent, i.e., a compoundof the invention (alone or in combination with another pharmaceuticalagent), to a patient, or application or administration of a therapeuticagent to an isolated tissue or cell line from a patient (e.g., fordiagnosis or ex vivo applications), who has HBV infection, a symptom ofHBV infection or the potential to develop HBV infection, with thepurpose to heal, alleviate, relieve, alter, remedy, ameliorate, improveor affect HBV infection, the symptoms of HBV infection or the potentialto develop HBV infection. Such treatments may be specifically tailoredor modified, based on knowledge obtained from the field ofpharmacogenomics.

As used herein, the term “prevent” or “prevention” means no disorder ordisease development if none had occurred, or no further disorder ordisease development if there had already been development of thedisorder or disease. Also considered is the ability of one to preventsome or all of the symptoms associated with the disorder or disease.

As used herein, the term “patient,” “individual” or “subject” refers toa human or a non-human mammal. Non-human mammals include, for example,livestock and pets, such as ovine, bovine, porcine, canine, feline andmurine mammals. Preferably, the patient, subject or individual is human.

As used herein, the terms “effective amount,” “pharmaceuticallyeffective amount” and “therapeutically effective amount” refer to anontoxic but sufficient amount of an agent to provide the desiredbiological result. That result may be reduction and/or alleviation ofthe signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system. An appropriate therapeutic amount inany individual case may be determined by one of ordinary skill in theart using routine experimentation.

As used herein, the term “pharmaceutically acceptable salts” refers toderivatives of the disclosed compounds wherein the parent compound ismodified by converting an existing acid or base moiety to its salt form.Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. The pharmaceutically acceptable salts of thepresent invention include the conventional non-toxic salts of the parentcompound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), eachof which is incorporated herein by reference in its entirety.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations. As used herein, “pharmaceuticallyacceptable carrier” also includes any and all coatings, antibacterialand antifungal agents, and absorption delaying agents, and the like thatare compatible with the activity of the compound useful within theinvention, and are physiologically acceptable to the patient.Supplementary active compounds may also be incorporated into thecompositions. The term “pharmaceutically acceptable carrier” may furtherinclude a pharmaceutically acceptable salt of the compound useful withinthe invention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

As used herein, the term “composition” or “pharmaceutical composition”refers to a mixture of at least one compound useful within the inventionwith a pharmaceutically acceptable carrier. The pharmaceuticalcomposition facilitates administration of the compound to a patient orsubject. Multiple techniques of administering a compound exist in theart including, but not limited to, intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

As used herein, the term “alkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a straight or branched chainhydrocarbon having the number of carbon atoms designated (i.e., C₁₋₆means one to six carbon atoms) and includes straight, branched chain, orcyclic substituent groups. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, andcyclopropylmethyl. Most preferred is (C₁-C₆)alkyl, particularly ethyl,methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

As used herein, the term “heteroalkyl” by itself or in combination withanother term means, unless otherwise stated, a stable straight orbranched chain alkyl group consisting of the stated number of carbonatoms and one or two heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may be optionallyoxidized and the nitrogen heteroatom may be optionally quaternized. Theheteroatom(s) may be placed at any position of the heteroalkyl group,including between the rest of the heteroalkyl group and the fragment towhich it is attached, as well as attached to the most distal carbon atomin the heteroalkyl group. Examples include: —O—CH₂—CH₂—CH₃,—CH₂—CH₂—CH₂—OH, —CH₂—CH₂—NH—CH₃, —CH₂—S—CH₂—CH₃, and —CH₂CH₂—S(═O)—CH₃.Up to two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃, or —CH₂—CH₂—S—S—CH₃. Preferred heteroalkyl groups have1-10 carbons.

As used herein, the term “alkoxy” employed alone or in combination withother terms means, unless otherwise stated, an alkyl group having thedesignated number of carbon atoms, as defined above, connected to therest of the molecule via an oxygen atom, such as, for example, methoxy,ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs andisomers. Preferred are (C₁-C₃) alkoxy, particularly ethoxy and methoxy.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine,more preferably, fluorine or chlorine.

As used herein, the term “cycloalkyl” refers to a mono cyclic orpolycyclic non-aromatic radical, wherein each of the atoms forming thering (i.e., skeletal atoms) is a carbon atom. In one embodiment, thecycloalkyl group is saturated or partially unsaturated. In anotherembodiment, the cycloalkyl group is fused with an aromatic ring.Cycloalkyl groups include groups having from 3 to 10 ring atoms.Illustrative examples of cycloalkyl groups include, but are not limitedto, the following moieties:

Monocyclic cycloalkyls include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.Dicyclic cycloalkyls include, but are not limited to,tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycycliccycloalkyls include adamantine and norbornane. The term cycloalkylincludes “unsaturated nonaromatic carbocyclyl” or “nonaromaticunsaturated carbocyclyl” groups, both of which refer to a nonaromaticcarbocycle as defined herein, which contains at least one carbon carbondouble bond or one carbon carbon triple bond.

As used herein, the term “heterocycloalkyl” or “heterocyclyl” refers toa heteroalicyclic group containing one to four ring heteroatoms eachselected from O, S and N. In one embodiment, each heterocycloalkyl grouphas from 4 to 10 atoms in its ring system, with the proviso that thering of said group does not contain two adjacent O or S atoms. Inanother embodiment, the heterocycloalkyl group is fused with an aromaticring. In one embodiment, the nitrogen and sulfur heteroatoms may beoptionally oxidized, and the nitrogen atom may be optionallyquaternized. The heterocyclic system may be attached, unless otherwisestated, at any heteroatom or carbon atom that affords a stablestructure. A heterocycle may be aromatic or non-aromatic in nature. Inone embodiment, the heterocycle is a heteroaryl.

An example of a 3-membered heterocycloalkyl group includes, and is notlimited to, aziridine. Examples of 4-membered heterocycloalkyl groupsinclude, and are not limited to, azetidine and a beta lactam. Examplesof 5-membered heterocycloalkyl groups include, and are not limited to,pyrrolidine, oxazolidine and thiazolidinedione. Examples of 6-memberedheterocycloalkyl groups include, and are not limited to, piperidine,morpholine and piperazine. Other non-limiting examples ofheterocycloalkyl groups are:

Examples of non-aromatic heterocycles include monocyclic groups such asaziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane,2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran,1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin, and hexamethyleneoxide.

As used herein, the term “aromatic” refers to a carbocycle orheterocycle with one or more polyunsaturated rings and having aromaticcharacter, i.e., having (4n+2) delocalized π (pi) electrons, where n isan integer.

As used herein, the term “aryl,” employed alone or in combination withother terms, means, unless otherwise stated, a carbocyclic aromaticsystem containing one or more rings (typically one, two or three rings),wherein such rings may be attached together in a pendent manner, such asa biphenyl, or may be fused, such as naphthalene. Examples of arylgroups include phenyl, anthracyl, and naphthyl. Preferred examples arephenyl and naphthyl, most preferred is phenyl.

As used herein, the term “heteroaryl” or “heteroaromatic” refers to aheterocycle having aromatic character. A polycyclic heteroaryl mayinclude one or more rings that are partially saturated. Examples includethe following moieties:

Examples of heteroaryl groups also include pyridyl, pyrazinyl,pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl,furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl,oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and1,3,4-oxadiazolyl.

Examples of polycyclic heterocycles and heteroaryls include indolyl(particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl,tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl),1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2-and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl,1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl,benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl),2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl(particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl,benzimidazolyl (particularly 2-benzimidazolyl), benzotriazolyl,thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, andquinolizidinyl.

Compounds of the Invention

The present invention relates to the discovery of compounds that areuseful in the treatment and prevention of HBV infection in man. In oneaspect, the compounds of the invention are useful in HBV treatment bydisrupting, accelerating, reducing, delaying or inhibiting normal HBVviral replication, thereby inducing aberrant viral replication andleading to antiviral effects such as disruption of virion assembly ordisassembly, or virion maturation, or virus egress.

The viral replication disruptors disclosed herein may be used asmonotherapy or in novel cross-class combination regimens for treatingHBV infection in man. Combination therapy with drugs exhibitingdifferent mechanism of action (MOA) that act at different steps in thevirus life cycle may deliver greater efficacy due to additive orsynergistic antiviral effects. Clinically evaluated HIV treatmentregimens have shown that combination therapy improves the efficacy ofviral load reduction, and dramatically reduces emergence of antiviralresistance. Combination therapy for the treatment of Hepatitis C (HCV)virus infection has also resulted in significant improvement insustained antiviral response and eradication rates. Thus, use of the HBVviral replication inhibitors of the present invention in combinationwith, for example, NA drugs, is likely to deliver a more profoundantiviral effect and greater disease eradication rates than currentstandards of care.

In one aspect, drug resistance poses a major threat to current therapiesfor chronic HBV infection, and cross-class combination therapy is aproven strategy for delaying emergence of drug resistance strains. Theviral replication disruptors of the present invention can, whenadministered alone or in combination with other HBV therapy, offerenhanced drug resistant profiles and improved management of chronic HBV.

The compounds useful within the invention may be synthesized usingtechniques well-known in the art of organic synthesis. The startingmaterials and intermediates required for the synthesis may be obtainedfrom commercial sources or synthesized according to methods known tothose skilled in the art.

In one aspect, the compound of the invention is a compound of Formula I:

or a pharmaceutically acceptable salt thereof;

wherein

three of G¹⁻⁴ are independently selected from C and CH, and one of G¹⁻⁴is N;

each R¹ is independently selected from H, halo, and CN;

each R² is, independently for each occurrence, selected from halo,C₁₋₆-alkyl, C₁₋₆-alkoxy, halo-(C₁₋₆-alkyl), di-halo-(C₁₋₆-alkyl), andtri-halo-(C₁₋₆-alkyl);

R³ is halo;

R⁴ is selected from C₁₋₆-alkyl, C₁₋₆-heteroalkyl, C₃₋₇-cycloalkyl,(C₁₋₆-alkyl)-(C₃₋₇-cycloalkyl), aryl, (C₁₋₆-alkyl)-aryl,C₃₋₇-heterocycloalkyl, (C₁₋₆-alkyl)-(C₃₋₇-heterocycloalkyl), heteroaryl,(C₁₋₆-alkyl)-C(O)O—(C₁₋₆-alkyl), and (C₁₋₆-alkyl)-heteroaryl, all ofwhich may be optionally substituted with halo, OH, CN, C₁₋₆-alkyl,O—(C₁₋₆-alkyl), CF₃, benzyl, C(O)OH, (C₁₋₆-alkyl)-C(O)OH, orC(O)O—(C₁₋₆-alkyl); and

n is 0, 1, or 2.

In one embodiment, ring A is

In another embodiment, ring A is

In still another embodiment, R⁴ is selected from C₁₋₆-alkyl,C₃₋₇-cycloalkyl, (C₁₋₆-alkyl)-(C₃₋₇-cycloalkyl), C₃₋₇-heterocycloalkyl,and (C₁₋₆-alkyl)-(C₃₋₇-heterocycloalkyl), all of which may be optionallysubstituted with OH or C₁₋₆-alkyl.

In an embodiment, the compound of Formula I is a compound of Formula Ia:

or a pharmaceutically acceptable salt thereof;

wherein

R¹ is selected from halo, and CN;

R³ is halo; and

R⁴ is selected from C₁₋₆-alkyl, C₁₋₆-heteroalkyl, C₃₋₇-cycloalkyl,(C₁₋₆-alkyl)-(C₃₋₇-cycloalkyl), aryl, (C₁₋₆-alkyl)-aryl,C₃₋₇-heterocycloalkyl, (C₁₋₆-alkyl)-(C₃₋₇-heterocycloalkyl), heteroaryl,(C₁₋₆-alkyl)-C(O)O—(C₁₋₆-alkyl), and (C₁₋₆-alkyl)-heteroaryl, all ofwhich may be optionally substituted with halo, OH, CN, C₁₋₆-alkyl,O—(C₁₋₆-alkyl), CF₃, benzyl, C(O)OH, (C₁₋₆-alkyl)-C(O)OH, orC(O)O—(C₁₋₆-alkyl).

In an embodiment of Formula Ia, R³ is F.

In another embodiment, R⁴ is selected from C₁₋₆-alkyl, C₃₋₇-cycloalkyl,(C₁₋₆-alkyl)-(C₃₋₇-cycloalkyl), (C₁₋₆-alkyl)-aryl,C₃₋₇-heterocycloalkyl, and (C₁₋₆-alkyl)-(C₃₋₇-heterocycloalkyl), all ofwhich may be optionally substituted with OH or C₁₋₆-alkyl.

In yet another embodiment, R⁴ is selected from C₁₋₆-alkyl,C₃₋₇-cycloalkyl, (C₁₋₆-alkyl)-(C₃₋₇-cycloalkyl), and (C₁₋₆-alkyl)-aryl,all of which may be optionally substituted with C₁₋₆-alkyl.

In still another embodiment, R⁴ is selected from C₁₋₆-alkyl,C₃₋₇-cycloalkyl, (C₁₋₆-alkyl)-(C₃₋₇-cycloalkyl), and (C₁₋₆-alkyl)-aryl,wherein the C₃₋₇-cycloalkyl and (C₁₋₆-alkyl)-aryl are substituted withC₁₋₆-alkyl.

In another embodiment of Formula Ia, or a pharmaceutically acceptablesalt thereof, R¹ is halo; R³ is halo; and R⁴ is selected fromC₁₋₆-alkyl, C₁₋₆-heteroalkyl, C₃₋₇-cycloalkyl, (C₁₋₆-alkyl)-(C₃₋₇-cycloalkyl), (C₁₋₆-alkyl)-aryl, C₃₋₇-heterocycloalkyl,(C₁₋₆-alkyl)-(C₃₋₇-heterocycloalkyl), heteroaryl, all of which may besubstituted with OH or C₁₋₆-alkyl.

In another embodiment of Formula Ia, or a pharmaceutically acceptablesalt thereof, R¹ is halo; R³ is halo; and R⁴ is selected fromC₁₋₆-alkyl, C₃₋₇-cycloalkyl, (C₁₋₆-alkyl)-(C₃₋₇-cycloalkyl), and(C₁₋₆-alkyl)-aryl, wherein the C₃₋₇-cycloalkyl and (C₁₋₆-alkyl)-arylgroups are optionally substituted with C₁₋₆-alkyl.

In another embodiment, R⁴ is selected from C₂₋₆-alkyl, C₃₋₇-cycloalkyl,(C₁₋₆-alkyl)-(C₃₋₇-cycloalkyl), and (C₁₋₆-alkyl)-aryl, wherein theC₃₋₇-cycloalkyl and (C₁₋₆-alkyl)-aryl groups are optionally substitutedwith C₁₋₆-alkyl.

In another embodiment, R⁴ is selected from C₁₋₆-alkyl, C₃₋₇-cycloalkyl,(C₁₋₆-alkyl)-(C₃₋₇-cycloalkyl), and (C₁₋₆-alkyl)-aryl, wherein theC₃₋₇-cycloalkyl and (C₁₋₆-alkyl)-aryl groups are substituted withC₁₋₆-alkyl.

In another embodiment, R⁴ is selected from C₂₋₆-alkyl, C₃₋₇-cycloalkyl,(C₁₋₆-alkyl)-(C₃₋₇-cycloalkyl), and (C₁₋₆-alkyl)-aryl, wherein theC₃₋₇-cycloalkyl and (C₁₋₆-alkyl)-aryl groups are substituted withC₁₋₆-alkyl.

In another embodiment of Formula Ia, or a pharmaceutically acceptablesalt thereof, the compound is selected from the group consisting of:

In another aspect, the compound of the invention is a compound ofFormula II:

wherein

each R¹ is independently selected from H, halo, CN, C₁₋₆-alkyl,C₁₋₆-alkoxy, halo-(C₁₋₆-alkyl), di-halo-(C₁₋₆-alkyl),tri-halo-(C₁₋₆-alkyl), OCF₃, N(H)S(O)₂—(C₁₋₆-alkyl), S(O)₂—(C₁₋₆-alkyl),C(H)(C₁₋₆-alkyl)OH, C(C₁₋₆-alkyl)₂OH, C(O)—(C₁₋₆-alkyl), andC₃₋₇-cycloalkyl;

R² is selected from halo, C₁₋₆-alkyl, C₁₋₆-alkoxy, halo-(C₁₋₆-alkyl),di-halo-(C₁₋₆-alkyl), tri-halo-(C₁₋₆-alkyl), (C₁₋₆-alkyl)-OH, C(O)OH,OCF₃, and C(O)O(C₁₋₆-alkyl);

R³ is halo;

or R³ and one R¹, together with the atoms to which they are attached,form an isobenzofuranone;

R⁴ is selected from C₁₋₆-heteroalkyl, C₃₋₇-heterocycloalkyl,(C₁₋₆-alkyl)-(C₃₋₇-heterocycloalkyl), heteroaryl, and(C₁₋₆-alkyl)-heteroaryl, all of which may be optionally independentlysubstituted with one or two of OH and C₁₋₆-alkyl;

or R⁴ is C₃₋₇-heterocycloalkyl optionally substituted with OH, whereinthe heteroatom is the group S(O)₂;

R⁵ is selected from H and C₁₋₆-alkyl; and

n is 0 or 1.

In one embodiment of Formula II, each R¹ is independently selected fromH, halo, CN, C₁₋₆-alkyl, CH₂F, CHF₂, CF₃, and C₃₋₇-cycloalkyl. Inanother embodiment, R² is selected from halo, C₁₋₆-alkyl,(C₁₋₆-alkyl)-OH, C(O)OH, and OCF₃. In still another embodiment, R⁴ isselected from C₁₋₆-heteroalkyl, C₃₋₇-heterocycloalkyl, and heteroaryl,all of which may be optionally independently substituted with one or twoof C₁₋₆-alkyl.

In another embodiment, R³ is F.

In another embodiment of Formula II, each R¹ is independently selectedfrom H, halo, and C₁₋₆-alkyl.

In yet another embodiment, one R¹ is H, and one R¹ is selected from H,halo, and C₁₋₆-alkyl.

In still another embodiment, R² is halo.

In another embodiment, R⁴ is selected from C₁₋₆-heteroalkyl, andheteroaryl, each of which may be optionally independently substitutedwith one or two of C₁₋₆-alkyl.

In yet another embodiment, n is 1.

In another embodiment of Formula II, or a pharmaceutically acceptablesalt thereof, each R¹ is independently selected from H, halo, andC₁₋₆-alkyl; R² is halo; R³ is halo; R⁴ is C₁₋₆-heteroalkyl orheteroaryl, each of which may be independently substituted with one ortwo of C₁₋₆-alkyl; R⁵ is H; and n is 0 or 1.

In another embodiment of Formula II, or a pharmaceutically acceptablesalt thereof, the compound is selected from the group consisting of:

In still another aspect, the compound of the invention is a compound ofFormula III:

wherein

ring A is heteroaryl;

two of G¹⁻⁴ are N and two of G¹⁻⁴ are CH;

each R¹ is independently selected from H and halo;

R³ is halo; and

R⁴ is C₃₋₇-cycloalkyl.

In one embodiment, ring A is

It will be appreciated that the description of the present inventionherein should be construed in congruity with the laws and principals ofchemical bonding. In some instances it may be necessary to remove ahydrogen atom in order to accommodate a substitutent at any givenlocation.

Preferred embodiments of Formula I, Formula Ia, Formula II, and FormulaIII, including pharmaceutically acceptable salts thereof, are shownbelow in Table 1 and are also considered to be “compounds of theinvention.” Some compounds of Table 1 do not include hydrogens onhydroxyl groups; it is understood that “—O” indicates a hydroxylsubstituent at these positions.

TABLE 1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

The invention further includes a composition comprising a compoundaccording to Formula I, Formula Ia, Formula II, or Formula III, or asalt, solvate, or N-oxide thereof. In one embodiment, the composition ispharmaceutical and further comprises at least one pharmaceuticallyacceptable carrier.

Compounds described herein also include isotopically-labeled compoundswherein one or more atoms is replaced by an atom having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number usually found in nature. Examples of isotopes suitablefor inclusion in the compounds described herein include and are notlimited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³²P, and ³⁵S. In one embodiment, isotopically-labeledcompounds are useful in drug and/or substrate tissue distributionstudies. In another embodiment, substitution with heavier isotopes suchas deuterium affords greater metabolic stability (for example, increasedin vivo half-life or reduced dosage requirements). In yet anotherembodiment, substitution with positron emitting isotopes, such as ¹¹C,¹⁸F, ¹⁵O and ¹³N, is useful in Positron Emission Topography (PET)studies for examining substrate receptor occupancy. Isotopically-labeledcompounds are prepared by any suitable method or by processes using anappropriate isotopically-labeled reagent in place of the non-labeledreagent otherwise employed.

In one embodiment, the compounds described herein are labeled by othermeans, including, but not limited to, the use of chromophores orfluorescent moieties, bioluminescent labels, or chemiluminescent labels.

The compounds described herein, and other related compounds havingdifferent substituents are synthesized using techniques and materialsdescribed herein and as described, for example, in Fieser and Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive OrganicTransformations (VCH Publishers Inc., 1989), March, Advanced OrganicChemistry 4^(th) Ed., (Wiley 1992); Carey and Sundberg, Advanced OrganicChemistry 4th Ed., Vols. A and B (Plenum 2000, 2001), and Green andWuts, Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (allof which are incorporated by reference for such disclosure). Generalmethods for the preparation of compound as described herein are modifiedby the use of appropriate reagents and conditions, for the introductionof the various moieties found in the formula as provided herein.

Compounds described herein are synthesized using any suitable proceduresstarting from compounds that are available from commercial sources, orare prepared using procedures described herein.

Methods of Treatment

The invention includes a method of treating an HBV infection in anindividual in need thereof, comprising administering to the individual atherapeutically effective amount of a compound of the invention.

The invention also includes a method of reducing the viral loadassociated with an HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound of the invention.

The invention further includes a method of reducing reoccurrence of anHBV infection in an individual in need thereof, comprising administeringto the individual a therapeutically effective amount of a compound ofthe invention.

The invention also includes a method of reducing an adversephysiological impact of an HBV infection in an individual in needthereof, comprising administering to the individual a therapeuticallyeffective amount of a compound of the invention.

The invention further includes a method of inducing remission of hepaticinjury from an HBV infection in an individual in need thereof,comprising administering to the individual a therapeutically effectiveamount of a compound of the invention.

The invention also includes a method of reducing the physiologicalimpact of long-term antiviral therapy for HBV infection in an individualin need thereof, comprising administering to the individual atherapeutically effective amount of a compound of the invention.

The invention further includes a method of prophylactically treating anHBV infection in an individual in need thereof, wherein the individualis afflicted with a latent HBV infection, comprising administering tothe individual a therapeutically effective amount of a compound of theinvention.

In one embodiment, the methods described herein comprise administeringto the individual at least one additional therapeutic agent selectedfrom the group consisting of an HBV vaccine, HBV polymerase inhibitor,interferon, pegylated interferon, viral entry inhibitor, viralmaturation inhibitor, capsid assembly modulator, reverse transcriptaseinhibitor, a TLR-agonist, and agents of distinct or unknown mechanism,and a combination thereof. In another embodiment, the pegylatedinterferon is pegylated interferon alpha (IFN-α), pegylated interferonlambda (IFN-k), or pegylated interferon gamma (IFN-γ). In still anotherembodiment, the reverse transcriptase inhibitor is at least one ofZidovudine, Didanosine, Zalcitabine, ddA, Stavudine, Lamivudine,Abacavir, Emtricitabine, Entecavir, Apricitabine, Atevirapine,ribavirin, acyclovir, famciclovir, valacyclovir, ganciclovir,valganciclovir, Tenofovir, Adefovir, cidofovir, Efavirenz, Nevirapine,Delavirdine, or Etravirine. In yet another embodiment, the compound andthe at least one additional therapeutic agent are co-formulated. Instill another embodiment, the compound and the at least one additionaltherapeutic agent are co-administered.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound of FormulaI, or a pharmaceutically acceptable salt thereof. In another embodiment,provided herein is a method of treating an HBV infection in anindividual in need thereof, comprising administering to the individual atherapeutically effective amount of compound of Formula Ia, or apharmaceutically acceptable salt thereof. In another embodiment,provided herein is a method of treating an HBV infection in anindividual in need thereof, comprising administering to the individual atherapeutically effective amount of compound of Formula II, or apharmaceutically acceptable salt thereof. In another embodiment,provided herein is a method of treating an HBV infection in anindividual in need thereof, comprising administering to the individual atherapeutically effective amount of compound of Formula III, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 2, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 6, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 8, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 9, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 11, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 28, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 41, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 58, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 59, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 66, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 67, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 72, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 73, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 75, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 76, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 77, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 78, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 79, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 80, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 81, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 82, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 83, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 84, or apharmaceutically acceptable salt thereof.

In another embodiment, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising administering tothe individual a therapeutically effective amount of compound 85, or apharmaceutically acceptable salt thereof.

Combination Therapies

The compounds of the present invention are intended to be useful incombination with one or more additional compounds useful for treatingHBV infection. These additional compounds may comprise compounds of thepresent invention or compounds known to treat, prevent, or reduce thesymptoms or effects of HBV infection. Such compounds include but are notlimited to HBV polymerase inhibitors, interferons, viral entryinhibitors, viral maturation inhibitors, literature-described capsidassembly modulators, and other agents with distinct or unknownmechanisms that affect the HBV life cycle and/or affect the consequencesof HBV infection.

In non-limiting examples, the compounds of the invention may be used incombination with one or more drugs (or a salt, solvate or prodrugthereof) selected from the group consisting of:

HBV reverse transcriptase inhibitors, and DNA and RNA polymeraseinhibitors, including but are not limited to: lamivudine (3TC, Zeffix,Heptovir, Epivir, and Epivir-HBV), entecavir (Baraclude, Entavir),adefovir dipivoxil (Hepsara, Preveon, bis-POM PMEA), tenofovirdisoproxil fumarate (Viread, TDF or PMPA);

interferons, including but not limited to interferon alpha (IFN-α),interferon lambda (IFN-λ), and interferon gamma (IFN-γ);

viral entry inhibitors;

viral maturation inhibitors;

literature-described capsid assembly modulators, such as but not limitedto BAY 41-4109;

compounds of distinct or unknown mechanism, such as but not limited toAT-61((E)-N-(1-chloro-3-oxo-1-phenyl-3-(piperidin-1-yl)prop-1-en-2-yl)benzamide),AT-130((E)-N-(1-bromo-1-(2-methoxyphenyl)-3-oxo-3-(piperidin-1-yl)prop-1-en-2-yl)-4-nitrobenzamide),and similar analogs.

In another embodiment, the additional therapeutic agent selected fromimmune modulator or immune stimulator therapies, which includesbiological agents belonging to the interferon class, such as interferonalpha 2a or 2b or modified interferons such as pegylated interferon,alpha 2a, alpha 2b, lamda; or TLR modulators such as TLR-7 agonists orTLR-9 agonists, or antiviral agents that block viral entry or maturationor target the HBV polymerase such as nucleoside or nucleotide ornon-nucleos(t)ide polymerase inhibitors, and agents of distinct orunknown mechanism including agents that disrupt the function of otheressential viral protein(s) or host proteins required for HBV replicationor persistence.

In an embodiment of the combination therapy, the reverse transcriptaseinhibitor and/or DNA and/or RNA polymerase inhibitor Zidovudine,Didanosine, Zalcitabine, ddA, Stavudine, Lamivudine, Abacavir,Emtricitabine, Entecavir, Apricitabine, Atevirapine, ribavirin,acyclovir, famciclovir, valacyclovir, ganciclovir, valganciclovir,Tenofovir, Adefovir, cidofovir, Efavirenz, Nevirapine, Delavirdine, orEtravirine.

In another embodiment of the combination therapy, the TLR-7 agonist isselected from the group consisting of SM360320(9-benzyl-8-hydroxy-2-(2-methoxy-ethoxy)adenine) and AZD 8848 (methyl[3-({[3-(6-amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-9-yl)propyl][3-(4-morpholinyl)propyl]amino}methyl)phenyl]acetate).

A synergistic effect may be calculated, for example, using suitablemethods such as, for example, the Sigmoid-E_(max) equation (Holford &Scheiner, 19981, Clin. Pharmacokinet. 6: 429-453), the equation of Loeweadditivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.Enzyme Regul. 22: 27-55). Each equation referred to above may be appliedto experimental data to generate a corresponding graph to aid inassessing the effects of the drug combination. The corresponding graphsassociated with the equations referred to above are theconcentration-effect curve, isobologram curve and combination indexcurve, respectively.

Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the patienteither prior to or after the onset of a HBV infection. Further, severaldivided dosages, as well as staggered dosages may be administered dailyor sequentially, or the dose may be continuously infused, or may be abolus injection. Further, the dosages of the therapeutic formulationsmay be proportionally increased or decreased as indicated by theexigencies of the therapeutic or prophylactic situation.

Administration of the compositions of the present invention to apatient, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto treat HBV infection in the patient. An effective amount of thetherapeutic compound necessary to achieve a therapeutic effect may varyaccording to factors such as the state of the disease or disorder in thepatient; the age, sex, and weight of the patient; and the ability of thetherapeutic compound to treat HBV infection in the patient. Dosageregimens may be adjusted to provide the optimum therapeutic response.For example, several divided doses may be administered daily or the dosemay be proportionally reduced as indicated by the exigencies of thetherapeutic situation. A non-limiting example of an effective dose rangefor a therapeutic compound of the invention is from about 1 and 5,000mg/kg of body weight/per day. One of ordinary skill in the art would beable to study the relevant factors and make the determination regardingthe effective amount of the therapeutic compound without undueexperimentation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

In particular, the selected dosage level will depend upon a variety offactors including the activity of the particular compound employed, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds or materials used incombination with the compound, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of HBV infection in a patient.

In one embodiment, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Inone embodiment, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a compound of theinvention and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol,in the composition. Prolonged absorption of the injectable compositionsmay be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate or gelatin. In oneembodiment, the pharmaceutically acceptable carrier is not DMSO alone.

In one embodiment, the compositions of the invention are administered tothe patient in dosages that range from one to five times per day ormore. In another embodiment, the compositions of the invention areadministered to the patient in range of dosages that include, but arenot limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It will be readily apparent toone skilled in the art that the frequency of administration of thevarious combination compositions of the invention will vary fromindividual to individual depending on many factors including, but notlimited to, age, disease or disorder to be treated, gender, overallhealth, and other factors. Thus, the invention should not be construedto be limited to any particular dosage regime and the precise dosage andcomposition to be administered to any patient will be determined by theattending physical taking all other factors about the patient intoaccount.

Compounds of the invention for administration may be in the range offrom about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg toabout 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg toabout 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80mg to about 500 mg, and any and all whole or partial incrementstherebetween.

In some embodiments, the dose of a compound of the invention is fromabout 1 mg and about 2,500 mg. In some embodiments, a dose of a compoundof the invention used in compositions described herein is less thanabout 10,000 mg, or less than about 8,000 mg, or less than about 6,000mg, or less than about 5,000 mg, or less than about 3,000 mg, or lessthan about 2,000 mg, or less than about 1,000 mg, or less than about 500mg, or less than about 200 mg, or less than about 50 mg. Similarly, insome embodiments, a dose of a second compound (i.e., a drug used fortreating Parkinson's Disease) as described herein is less than about1,000 mg, or less than about 800 mg, or less than about 600 mg, or lessthan about 500 mg, or less than about 400 mg, or less than about 300 mg,or less than about 200 mg, or less than about 100 mg, or less than about50 mg, or less than about 40 mg, or less than about 30 mg, or less thanabout 25 mg, or less than about 20 mg, or less than about 15 mg, or lessthan about 10 mg, or less than about 5 mg, or less than about 2 mg, orless than about 1 mg, or less than about 0.5 mg, and any and all wholeor partial increments thereof.

In one embodiment, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound of the invention, aloneor in combination with a second pharmaceutical agent; and instructionsfor using the compound to treat, prevent, or reduce one or more symptomsof HBV infection in a patient.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents.

Routes of administration of any of the compositions of the inventioninclude oral, nasal, rectal, intravaginal, parenteral, buccal,sublingual or topical. The compounds for use in the invention may beformulated for administration by any suitable route, such as for oral orparenteral, for example, transdermal, transmucosal (e.g., sublingual,lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- andperivaginally), (intra)nasal and (trans)rectal), intravesical,intrapulmonary, intraduodenal, intragastrical, intrathecal,subcutaneous, intramuscular, intradermal, intra-arterial, intravenous,intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Dosing

The therapeutically effective amount or dose of a compound of thepresent invention will depend on the age, sex and weight of the patient,the current medical condition of the patient and the progression of HBVinfection in the patient being treated. The skilled artisan will be ableto determine appropriate dosages depending on these and other factors.

A suitable dose of a compound of the present invention may be in therange of from about 0.01 mg to about 5,000 mg per day, such as fromabout 0.1 mg to about 1,000 mg, for example, from about 1 mg to about500 mg, such as about 5 mg to about 250 mg per day. The dose may beadministered in a single dosage or in multiple dosages, for example from1 to 4 or more times per day. When multiple dosages are used, the amountof each dosage may be the same or different. For example, a dose of 1 mgper day may be administered as two 0.5 mg doses, with about a 12-hourinterval between doses.

It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, is reduced, as a function of theviral load, to a level at which the improved disease is retained. In oneembodiment, patients require intermittent treatment on a long-term basisupon any recurrence of symptoms and/or infection.

The compounds for use in the method of the invention may be formulatedin unit dosage form. The term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosage for patients undergoingtreatment, with each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect,optionally in association with a suitable pharmaceutical carrier. Theunit dosage form may be for a single daily dose or one of multiple dailydoses (e.g., about 1 to 4 or more times per day). When multiple dailydoses are used, the unit dosage form may be the same or different foreach dose.

Toxicity and therapeutic efficacy of such therapeutic regimens areoptionally determined in cell cultures or experimental animals,including, but not limited to, the determination of the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between the toxicand therapeutic effects is the therapeutic index, which is expressed asthe ratio between LD₅₀ and ED₅₀. Capsid assembly inhibitors exhibitinghigh therapeutic indices are preferred. The data obtained from cellculture assays and animal studies is optionally used in formulating arange of dosage for use in human. The dosage of such capsid assemblyinhibitors lies preferably within a range of circulating concentrationsthat include the ED₅₀ with minimal toxicity. The dosage optionallyvaries within this range depending upon the dosage form employed and theroute of administration utilized.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Example: Preparation of the Compounds of the Invention

FIGS. 1-3 show general schemes used to prepare selected compounds of theinvention.

Intermediate A: 2-Bromo-N-(3-chloro-4-fluorophenyl)isonicotinamide

To a stirring solution of 2-bromoisonicotinic acid (6.00 g, 29.70 mmol)in anhydrous DMF (100 mL) was added HATU (13.55 g, 35.64 mmol),3-chloro-4-fluoroaniline (4.54 g, 31.19 mmol), Hünigs base (15.56 mL,89.10 mmol) and a catalytic amount of DMAP. The resulting mixture wasstirred at rt for 16 h. The reaction was diluted with EtOAc and brine.The organic phase was then washed once more with brine, 0.5M HCl (2×),water and concentrated in vacuo. The resulting crude solid was suspendedin DCM, stirred for 20 min, and filtered. The subsequent solid affordedthe title compound in pure form. LC-MS: 330 (M+H)⁺.

Intermediate B: 2-Bromo-N-(3,4,5-trifluorophenyl)isonicotinamide

The title compound was prepared in an analogous manner to Intermediate Ausing 3,4,5-trifluoroaniline. LC-MS: 332 (M+H)⁺.

Intermediate C: 2-Bromo-N-(3,4-difluorophenyl)isonicotinamide

The title compound was prepared in an analogous manner to Intermediate Ausing 3,4-difluoroaniline. LC-MS: 314 (M+H)⁺.

Intermediate D: 2-Amino-N-(3-chloro-4-fluorophenyl)isonicotinamide

To a stirring solution of 2-aminoisonicotinic acid (5.00 g, 36.2 mmol)in anhydrous DMF (100 mL) was added HATU (16.5 g, 35.64 mmol),3-chloro-4-fluoroaniline (5.27 g, 36.2 mmol), diisopropylethylamine (19mL, 110 mmol) and dimethylaminopyridine (750 mg, 6.1 mmol). Theresulting mixture was stirred at RT for 16 hrs then diluted with EtOAcand saturated aqueous NaHCO₃. The organics were then washed with brine,dried over MgSO₄ and partially concentrated in vacuo. The resultingsolid that crashed out was filtered and found to be pure title compound.The filtrate was concentrated and purified by c18 silica gelchromatography, eluting with a gradient of 5% to 100% MeCN in watercontaining 0.5% formic acid. Product containing fractions wereconcentrated, diluted with saturated aqueous NaHCO₃ and extracted withEtOAc. The organics were then dried over MgSO₄ and concentrated in vacuoto afford additional title compound. LC-MS: 266 (M+H)⁺.

Intermediate E: 2-Amino-N-(3,4-difluorophenyl)isonicotinamide

Prepared in analogous fashion to Intermediate D using3,4-difluoroaniline.

Intermediate F: 2-(Cyclopropanesulfonamido)isonicotinic acid

Step 1;

To a solution of methyl 2-aminoisonicotinate (0.408 g, 2.68 mmol) inanhydrous pyridine (10 mL) was added dimethylaminopyridine (0.01 g, 0.08mmol) and cyclopropanesulfonyl chloride (0.27 mL, 2.7 mmol). Theresulting mixture was heated to 60° C. for 16 hrs. Volatiles wereremoved in vacuo and the residue was diluted with EtOAc and 1N HCl. Theorganics were washed with 1N HCl (2×), brine, dried over MgSO₄, filteredand concentrated in vacuo. The resulting crude material was purified onsilica gel eluting with a solvent gradient of 0% to 100% EtOAc inhexanes to afford methyl 2-(cyclopropanesulfonamido)isonicotinate.

Step 2;

To a solution of methyl 2-(cyclopropanesulfonamido)isonicotinate (0.400g, 1.56 mmol) in methanol (10 mL) and tetrahydrofuran (20 mL) was added3N NaOH (10 mL) and the resulting mixture was stirred for 1 h. Thereaction mixture was acidified with 1N HCl and extracted with EtOAc(2×). The combined organics were washed with brine, dried over MgSO₄ andthe volatiles were removed in vacuo to provide the title compound.LC-MS: 243 (M+H)⁺.

Intermediate G: 5-(Cyclopropanesulfonamido)nicotinic acid

The title compound was prepared in an analogous manner to Intermediate Fusing methyl 5-aminonicotinate as a starting material. LC-MS: 243(M+H)⁺.

Intermediate H: 6-(Cyclopropanesulfonamido)picolinic acid

The title compound was prepared in an analogous manner to Intermediate Fusing methyl 6-aminopicolinate as a starting material. LC-MS: 243(M+H)⁺.

Intermediate I: 3-amino-N-(3-chloro-4-fluorophenyl)benzamide

To a solution of 3-((tert-butoxycarbonyl)amino)benzoic acid (1 g, 4.21mmol) in DMF (10 mL) was added 3-chloro-4-fluoroaniline (613 mg, 4.21mmol), N,N-diisopropylethylamine (1.47 mL, 8.42 mmol) and HATU (1.76 g,4.63 mmol). Catalytic DMAP was added (˜20 mg) and the reaction mixturestirred at room temperature for 16 hrs. The solution was diluted withEtOAc and washed with 1N HCl, brine and the organic phase separated,dried (MgSO₄), filtered and evaporated in vacuo. The isolated solid waspurified using silica gel chromatography eluting with 0 to 40% EtOAc inhexanes to afford the intermediate tert-butyl(3-((3-chloro-4-fluorophenyl)carbamoyl)phenyl)carbamate as a colorlesssolid. This was dissolved in dichloromethane (15 mL) to which was addedTFA (5 mL) and the reaction mixture stirred at room temperature untilLCMS analysis determined complete reaction. The solution was thendiluted with EtOAc, quenched with the addition of saturated aqueousNaHCO₃, the organic phase separated, dried (MgSO₄), filtered andevaporated in vacuo to afford the title compound.

Compound 1:N-(3-Chloro-4-fluorophenyl)-6-(cyclopropanesulfonamido)picolinamide

The title compound was prepared in an analogous manner to Compound 2using Intermediate H as a starting material. LC-MS: 370 (M+H)⁺.

Compound 2:N-(3-Chloro-4-fluorophenyl)-2-(cyclopropanesulfonamido)isonicotinamide

To a mixture of copper(I) iodide (0.23 g, 1.21 mmol), potassiumcarbonate (1.68 g, 12.14 mmol) and cyclopropylsulfonamide (0.809 g, 6.67mmol) in a RBF under nitrogen atmosphere was added DMF (60 mL),trans-(1R,2R)—N,N′-bismethyl-1,2-cyclohexanediamine (0.344 g, 2.42 mmol)and 2-bromo-N-(3-chloro-4-fluorophenyl)isonicotinamide (Intermediate A)(2.00 g, 6.07 mmol). The resulting mixture was sub-surface purged withnitrogen gas for 10 minutes before heating to 100° C. for 16 h. Thereaction was cooled to RT and diluted with EtOAc and 0.5M HCl. Theorganics were washed twice with 0.5M HCl, water (2×) and concentrated invacuo. The resulting crude solid was suspended in DCM, stirred for 1 hrand filtered. The resulting solid afforded the title compound in pureform. LC-MS: 370 (M+H)⁺.

Compound 3:N-(3-Chloro-4-fluorophenyl)-5-(cyclopropanesulfonamido)nicotinamide

To a stirring solution of Intermediate G (0.077 g, 0.32 mmol) inanhydrous DMF (3 mL) was added HATU (0.150 g, 0.38 mmol),3-chloro-4-fluoroaniline (0.046 g, 0.32 mmol), diisopropylethylamine(0.17 mL, 0.95 mmol) and dimethylaminopyridine (0.01 g, 0.08 mmol). Theresulting mixture was stirred at RT for 4 h then diluted with EtOAc andwater. The organic layer was washed with brine, dried over MgSO₄ andconcentrated in vacuo. The residue was purified by silica gelchromatography eluting with a gradient of 0% to 100% EtOAc in hexanes toprovide the title compound. LC-MS: 370 (M+H)⁺.

Compound 4:N-(3-Chloro-4-fluorophenyl)-4-(cyclopropanesulfonamido)picolinamide

The title compound was prepared in an analogous manner to Compound 14using 2-bromopyridin-4-amine as a starting material. LC-MS: 370 (M+H)⁺.

Compound 5:2-(Cyclopropanesulfonamido)-N-(3,4,5-trifluorophenyl)isonicotinamide

The title compound was prepared in an analogous manner to Compound 3using 3,4,5-trifluoroaniline and Intermediate F as a starting materials.LC-MS: 372 (M+H)⁺.

Compound 6:2-(Cyclopropanesulfonamido)-N-(3,4-difluorophenyl)isonicotinamide

The title compound was prepared in an analogous manner to Compound 3using 3,4-difluoroaniline and Intermediate F as a starting materials.LC-MS: 354 (M+H)⁺.

Compound 7:N-(3-Cyano-4-fluorophenyl)-2-(cyclopropanesulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 3using 5-amino-2-fluorobenzonitrile and Intermediate F as startingmaterials. LC-MS: 361 (M+H)⁺.

Compound 8:N-(3-Chloro-4-fluorophenyl)-2-(1-methylcyclopropanesulfonamido)isonicotinamide

To a solution of Intermediate D (0.150 g, 0.57 mmol) in anhydrouspyridine (5 mL) was added dimethylaminopyridine (0.01 g, 0.08 mmol) and1-methylcyclopropane-1-sulfonyl chloride (0.081 mL, 0.68 mmol). Theresulting mixture was heated to 60° C. for 16 hrs. Volatiles wereremoved in vacuo and the residue was diluted with EtOAc and 1N HCl. Theorganics were washed with 1N HCl (2×), brine, dried over MgSO₄, filteredand concentrated in vacuo. The resulting crude material was purified onsilica gel eluting with a solvent gradient of 0% to 100% EtOAc inhexanes to provide the title compound. LC-MS: 384 (M+H)⁺.

Compound 9:N-(3-Chloro-4-fluorophenyl)-2-(1-methylcyclopropanesulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using ethanesulfonyl chloride as a starting material. LC-MS: 358 (M+H)⁺.

Compound 10:N-(3-Chloro-4-fluorophenyl)-2-(3,3,3-trifluoropropylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using 3,3,3-trifluoropropane-1-sulfonyl chloride as a starting material.LC-MS: 426 (M+H)⁺.

Compound 11:N-(3-Chloro-4-fluorophenyl)-2-(propylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using propane-1-sulfonyl chloride as a starting material. LC-MS: 372(M+H)⁺.

Compound 12:N-(3-Chloro-4-fluorophenyl)-2-(cyclohexanesulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using cyclohexanesulfonyl chloride as a starting material. LC-MS: 412(M+H)⁺.

Compound 13:N-(3-Chloro-4-fluorophenyl)-2-(4-methylphenylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using p-toluenesulfonyl chloride as a starting material. LC-MS: 420(M+H)⁺.

Compound 14:N-(3-Chloro-4-fluorophenyl)-2-(cyclopropanesulfonamido)-6-methylisonicotinamide

Step 1;

To a solution of 4-chloro-6-methylpyridin-2-amine (0.33 g, 2.34 mmol) inanhydrous pyridine (6 mL) at 0° C. was added dimethylaminopyridine(0.029 g, 0.23 mmol) and cyclopropanesulfonyl chloride (0.41 mL, 3.97mmol). The resulting mixture was stirred cold for 5 minutes then heatedto 60° C. for 16 h. The reaction was cooled to RT and diluted with EtOAcand 1:1 1M HCl brine. The organics were washed twice with 1M HCl, brine(2×), dried over Na₂SO₄, filtered and concentrated in vacuo. Theresulting crude material was purified on silica gel eluting with asolvent gradient of 0% to 70% EtOAc in hexanes to affordN-(4-chloro-6-methylpyridin-2-yl)cyclopropanesulfonamide.

Step 2;

Combined N-(4-chloro-6-methylpyridin-2-yl)cyclopropanesulfonamide (0.18g, 0.73 mmol), sodium carbonate (0.15 g, 1.46 mmol),3-chloro-4-fluoroaniline (0.16 g, 1.10 mmol), toluene (10 mL), xantphos(0.018 g, 0.03 mmol), and Pd(OAc)₂ (0.007 g, 0.03 mmol) in a RBF whichwas stirred and purged with carbon monoxide gas. The resulting mixturewas stirred under a carbon monoxide atmosphere and heated to 90° C. for16 h. The reaction was cooled to RT and diluted with EtOAc and brine.The organics were washed with water, dried over Na₂SO₄, filtered andconcentrated in vacuo. The resulting crude material was purified onsilica gel eluting with a solvent gradient of 10% to 100% EtOAc inhexanes to afford the title compound with impurities. The residue thusobtained was further purified using a preparatory TLC plate using 7%methanol in DCM as eluent to afford the title compound. LC-MS: 384(M+H)⁺.

Compound 15:N-(3-Chloro-4-fluorophenyl)-2-(cyclopropanesulfonamido)-6-fluoroisonicotinamide

Step 1;

To a solution of 2,6-difluoroisonicotinic acid (1.0 g, 6.3 mmol) in DMF(15 mL) was added HATU (2.87 g, 7.5 mmol) followed byN,N-diisopropylethylamine (3.3 mL, 18.9 mmol). After stirring at roomtemperature for 18 hrs, the reaction mixture was quenched with sat'd.aq. NH₄Cl solution, extracted with EtOAc, washed with water, dried(MgSO₄), filtered and evaporated in vacuo. The resulting material wastriturated from a mixture of EtOAc and hexanes to afford a colorlesssolid isolated via filtration, dried in vacuo and used without furtherpurification.

Step 2;

To a solution of the isolatedN-(3-chloro-4-fluorophenyl)-2,6-difluoroisonicotinamide (400 mg, 1.40mmol) in anhydrous DMSO (5 mL) was added K₂CO₃ (400 mg, 2.9 mmol) in asealable vial which was then closed and heated at 110° C. for 18 hrs.After cooling, the mixture was diluted with sat′d. aq NH₄Cl solution,extracted with EtOAc, the organic phase separated, washed with water,dried (MgSO₄), filtered and evaporated in vacuo. Purification via silicagel chromatography afforded the title compound as a colorless solid.LC-MS: 388 (M+H)⁺.

Compound 16:N-(3-Chloro-4-fluorophenyl)-2-fluoro-6-(phenylsulfonamido)isonicotinamide

Prepared in an analogous fashion to Compound 15 using benzenesulfonamidein step 2. LC-MS: 388 (M+H)⁺ 424.

Compound 17:N-(3-Chloro-4-fluorophenyl)-2-(phenylmethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using phenylmethanesulfonyl chloride as a starting material. LC-MS: 420(M+H)⁺.

Compound 18:N-(3-Chloro-4-fluorophenyl)-2-(2-methoxyethylsulfonamido)isonicotinamide

To a solution of 2-amino-N-(3-chloro-4-fluorophenyl)isonicotinamide(Intermediate D) (0.15 g, 0.57 mmol) in anhydrous dioxane (5 mL) wasadded triethylamine (0.16 mL, 1.13 mmol) and 2-methoxyethanesulfonylchloride (0.11 mL, 0.903 mmol). The resulting mixture was stirred andheated to 80 C for 48 h. The reaction was cooled to RT and evaporated invacuo. The organics were washed twice with 1M HCl, brine (2×), driedover Na₂SO₄, filtered and concentrated in vacuo. The resulting crudematerial was purified on silica gel eluting with a solvent gradient of10% to 100% EtOAc in hexanes to afford the title compound. LC-MS: 386(M+H)⁺.

Compound 19:N-(3-Chloro-4-fluorophenyl)-2-(thiophene-2-sulfonamido)isonicotinamide

To a solution of 2-amino-N-(3-chloro-4-fluorophenyl)isonicotinamide(Intermediate D) (0.15 g, 0.57 mmol) in anhydrous pyridine (5 mL) wasadded thiophene-2-sulfonyl chloride (0.17 g, 0.903 mmol). The resultingmixture was stirred at RT for 16 h. The reaction was diluted with EtOAcand 1M HCl. The organics were washed twice with brine, water,coevaporated with heptanes and concentrated in vacuo. The product wascrashed out of 10% MeOH/DCM. The resulting filtered solid afforded thetitle compound. LC-MS: 412 (M+H)⁺.

Compound 20:N-(3-Chloro-4-fluorophenyl)-2-(cyclohexylmethylsulfonamido)isonicotinamide

To a solution of 2-amino-N-(3-chloro-4-fluorophenyl)isonicotinamide(Intermediate D) (0.15 g, 0.57 mmol) in anhydrous pyridine (5 mL) andcyclohexylmethanesulfonyl chloride (0.13 g, 0.79 mmol). The resultingmixture was stirred at RT for 16 h. The reaction was diluted with EtOAcand 1M HCl. The organics were washed twice with brine, water, andconcentrated in vacuo. The resulting crude material was purified onsilica gel eluting with a solvent gradient of 10% to 100% EtOAc inhexanes to afford the title compound. LC-MS: 426 (M+H)⁺.

Compound 21:N-(3-Chloro-4-fluorophenyl)-2-(2-phenylethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using 2-phenylethanesulfonyl chloride. LC-MS: 434 (M+H)⁺.

Compound 22:N-(3-Chloro-4-fluorophenyl)-2-(cyclopropanesulfonamido)-5-fluoroisonicotinamide

Step 1:

To a solution of 2-bromo-5-fluoroisonicotinic acid (3.0 g, 13.6 mmol) ina mixture of MeOH (10 mL) and benzene (20 mL) cooled to 0° C. was added(trimethylsilyl)diazomethane (2.0M solution in hexanes; 14 mL, 28 mmol)over a 10 minute period. After stirring at room temperature for 1.5 hrs,the solution was evaporated to dryness and the residue purified onsilica gel eluting with a gradient of 0 to 40% EtOAc in hexanes toafford methyl 2-bromo-5-fluoroisonicotinate as a colorless solid.

Step 2:

Cyclopropanesulfonamide (310 mg, 2.56 mmol), cesium carbonate (1.1 g,3.38 mmol), Pd₂(dba)₃ (40 mg, 0.044 mmol), Xantphos (51 mg, 0.09 mmol)and methyl 2-bromo-5-fluoroisonicotinate (500 mg, 2.14 mmol) were mixedin a sealable vial. To this mixture was added p-dioxane (10 mL) and thesuspension degassed via sub-surface sparging with nitrogen gas for 5minutes. The vial was then sealed and heated at 100° C. for 3 hoursafter which point the reaction mixture turned green to red. Aftercooling, the solution was partitioned between water and EtOAc, theaqueous acidified with 1N HCl and extracted with EtOAc (2×). The organicphases were combined, dried (MgSO₄), filtered and evaporated in vacuo.¹H NMR analysis confirmed isolation of methyl2-(cyclopropanesulfonamido)-5-fluoroisonicotinate as a colorless solid.

Step 3:

The title compound was prepared according to the same procedure outlinedfor Compound 3 using the previously isolated acid and purifying theresultant amide through trituration from acetone/hexanes. LC-MS: 388(M+H)⁺.

Compound 23:N-(3-Chloro-4-fluorophenyl)-2-(2-cyclopropylethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using 2-cyclopropylethanesulfonyl chloride as a starting material.LC-MS: 398 (M+H)⁺.

Compound 24:N-(3-Chloro-4-fluorophenyl)-2-((1-cyanocyclopropyl)methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using (1-cyanocyclopropyl)methanesulfonyl chloride as a startingmaterial. LC-MS: 409 (M+H)⁺.

Compound 25:N-(3-Chloro-4-fluorophenyl)-2-(3-methylbutylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using 3-methylbutane-1-sulfonyl chloride as a starting material. LC-MS:400 (M+H)⁺.

Compound 26:N-(3-Chloro-4-fluorophenyl)-2-(cyclopropanesulfonamido)-5-methoxyisonicotinamide

Step 1:

To a solution of the methyl ester isolated from Compound 22 Step 1 (1.0g, 4.27 mmol) in MeOH (10 mL) was added NaOMe (25% wt solution: 5 mL).After stirring at rt for 1.5 hrs, the reaction was quenched by additionof sat′d. aq. NH₄Cl solution. The resulting mixture was extracted withEtOAc, the organic phase separated, dried (MgSO₄), filtered andevaporated in vacuo. Purification via silica gel chromatography affordedmethyl 2-bromo-5-methoxyisonicotinate as a colorless solid.

Step 2:

tert-Butyl carbamate (300 mg, 2.56 mmol), cesium carbonate (1.1 g, 3.38mmol), Pd₂(dba)₃ (40 mg, 0.044 mmol), Xantphos (51 mg, 0.09 mmol) andmethyl 2-bromo-5-methoxyisonicotinate (527 mg, 2.14 mmol) were mixed ina sealable vial. To this mixture was added p-dioxane (10 mL) and thesuspension degassed via sub-surface sparging with nitrogen gas for 5minutes. The vial was then sealed and heated at 100° C. for 5 hoursafter which point the reaction mixture turned green to red. Aftercooling, the solution was partitioned between water and EtOAc, theaqueous acidified with 1N HCl and extracted with EtOAc (2×). The organicphases were combined, dried (MgSO₄), filtered and evaporated in vacuo.Purification of the isolated residue on silica gel eluting with 0 to 40%EtOAc in hexanes afforded methyl2-((tert-butoxycarbonyl)amino)-5-methoxyisonicotinate as a colorlesssolid

Step 3:

Methyl 2-((tert-butoxycarbonyl)amino)-5-methoxyisonicotinate (300 mg;1.06 mmol) was dissolved in 8 mL of 4N HCl in dioxane and stirred at rtfor 18 hrs. The reaction mixture was evaporated to dryness in vacuo toafford methyl 2-amino-5-methoxyisonicotinate, used without furtheranalysis or purification.

Step 4:

To a solution of the isolated methyl 2-amino-5-methoxyisonicotinate(1.06 mmol) in pyridine (4 mL) was added cyclopropanesulfonyl chloride(110 μL, 1.07 mmol) and the mixture stirred for 18 hrs. The mixture wasdiluted with EtOAc, washed with water, dried (MgSO₄), filtered andevaporated in vacuo. Purification of the residue on silica gel affordedmethyl 2-(cyclopropanesulfonamido)-5-methoxyisonicotinate as a colorlesssolid.

Step 5:

Methyl 2-(cyclopropanesulfonamido)-5-methoxyisonicotinate (190 mg, 0.66mmol) was dissolved in a mixture of THF (3 mL) and water (1 mL) to whichwas added LiOH.H₂O (80 mg, 1.9 mmol). After stirring at rt for 12 hrs,the solution was acidified with 1N HCl, extracted with EtOAc, theorganic phase separated, dried (MgSO₄), filtered and evaporated invacuo. The afforded 2-(cyclopropanesulfonamido)-5-methoxyisonicotinicacid was used in the final step without further purification.

Step 6:

2-(Cyclopropanesulfonamido)-5-methoxyisonicotinic acid (80.0 mg, 0.294mmol) was dissolved in DMF (3 mL) to which was added HATU (125.0 mg,0.324 mmol) followed by Hünigs base (0.3 mL, 1.7 mmol). After stirringfor 1 hr, 3-chloro-4-fluoroaniline (52 mg, 0.357 mmol) was added and thereaction mixture maintained at rt for 18 hrs. The solution was thendiluted with EtOAc and 1N HCl, the organic phase separated, washed withwater, dried (MgSO₄), filtered and evaporated in vacuo. The residue wastriturated with a mixture of EtOAc and hexanes to afford the titlecompound as a colorless solid. LC-MS: 400 (M+H)⁺.

Compound 27:5-Chloro-N-(3-chloro-4-fluorophenyl)-2-(cyclopropanesulfonamido)isonicotinamide

Step 1;

To a solution of methyl 2-amino-5-chloroisonicotinate (0.50 g, 2.68mmol) in anhydrous pyridine (10 mL) at 0° C. was added catalyticdimethylaminopyridine and cyclopropanesulfonyl chloride (0.44 mL, 4.29mmol). The resulting mixture was heated to 35° C. for 16 h. The reactionwas cooled to RT and diluted with EtOAc and brine. The organics werewashed with 1M HCl, brine, and water, coevaporated with heptanes andconcentrated in vacuo. The resulting crude material was purified on C₁₈column eluting with a solvent gradient of 20% to 100% acetonitrile inwater to afford methyl5-chloro-2-(cyclopropanesulfonamido)isonicotinate.

Step 2;

Combined methyl 5-chloro-2-(cyclopropanesulfonamido)isonicotinate (0.23g, 0.77 mmol), THF (6.2 mL), MeOH (6.2 mL) and 1 M LiOH (6.19 mL, 6.19mmol) and stirred at RT for 16 h. The reaction was concentrated anddiluted with EtOAc and 1M HCl. The organics were washed with water andconcentrated in vacuo. The resulting crude material containing5-chloro-2-(cyclopropanesulfonamido)isonicotinic acid was used as is inthe next step.

Step 3;

The title compound was prepared in an analogous manner to Intermediate Ausing HATU coupling conditions and5-chloro-2-(cyclopropanesulfonamido)isonicotinic acid. LC-MS: 405(M+H)⁺.

Compound 28:N-(3-Chloro-4-fluorophenyl)-2-(cyclopentylmethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using cyclopentylmethanesulfonyl chloride. LC-MS: 412 (M+H)⁺.

Compound 29:N-(3-Chloro-4-fluorophenyl)-2-((4-fluorophenyl)methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using (4-fluorophenyl)methanesulfonyl chloride as a starting material.LC-MS: 438 (M+H)⁺.

Compound 30:N-(3-Chloro-4-fluorophenyl)-2-(p-tolylmethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using (p-tolylmethanesulfonyl chloride as a starting material. LC-MS:434 (M+H)⁺.

Compound 31:N-(3,4-Difluorophenyl)-2-((4-fluorophenyl)methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using (p-tolylmethanesulfonyl chloride and Intermediate D as a startingmaterial. LC-MS: 434 (M+H)⁺.

Compound 32:N-(3-Chloro-4-fluorophenyl)-2-(1,1-dimethylethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 2using 2-methylpropane-2-sulfonamide as a starting material. LC-MS: 386(M+H)⁺.

Compound 33:N-(3-Chloro-4-fluorophenyl)-2-((1-hydroxycyclobutyl)methylsulfonamido)isonicotinamide

Step 1;

To a solution of benzylamine (4.00 g, 37.33 mmol) in DCM (50 mL) at 0°C. was added methanesulfonyl chloride (1.44 mL, 18.66 mmol) dropwise.The resulting mixture was stirred cold for 10 minutes then diluted withwater. The organics were washed with water, 1M HCl, dried over Na₂SO₄,filtered and concentrated in vacuo. The resulting crude materialcontaining N-benzylmethanesulfonamide was used as is in the next step.

Step 2;

To a solution of N-benzylmethanesulfonamide (1.00 g, 5.39 mmol) inanhydrous THF (10 mL) at −78° C. under a nitrogen atmosphere was addednBuLi (2.5M in hexanes, 4.32 mL, 10.78 mmol) dropwise. After 5 minutesof stirring at −78° C., cyclobutanone (1.92 mL, 21.56) was addeddropwise. The resulting solution was stirred at −78° C. for 2 h, RT 1hr, and quenched with 1 mL of acetic acid. The reaction was evaporatedand the residue was diluted with EtOAc and a saturated solution ofNaHCO₃. The organics were washed once more with a saturated solution ofNaHCO₃, brine, and water, and concentrated in vacuo. The resulting crudematerial was purified on silica gel eluting with a solvent gradient of0% to 9% MeOH in DCM to affordN-benzyl-1-(1-hydroxycyclobutyl)methanesulfonamide which wascontaminated with impurities. The resulting solid was triturated in 1:1EtOAc/heptanes and filtered to obtain pure material.

Step 3;

Combined N-benzyl-1-(1-hydroxycyclobutyl)methanesulfonamide (0.34 g,1.33 mmol), Pd(OH)₂ (70 mg, 20% wt/wt), MeOH (6.2 mL) and stirred undera hydrogen atmosphere at 60° C. for 16 h. The reaction was filteredthrough a pad of celite and concentrated in vacuo to obtain(1-hydroxycyclobutyl)methanesulfonamide which was used as is in the nextstep.

Step 4;

The title compound was prepared in an analogous manner to Compound 2using (1-hydroxycyclobutyl)methanesulfonamide and CuI couplingconditions. LC-MS: 414 (M+H)⁺.

Compound 34:N-(3-Chloro-4-fluorophenyl)-2-(2-hydroxy-2-methylpropylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to compound 33using acetone in step 2. LC-MS: 402 (M+H)⁺.

Compound 35:N-(3-Chloro-4-fluorophenyl)-2-(methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 2using methanesulfonamide and CuI coupling conditions. LC-MS: 402 (M+H)⁺.

Compound 36:N-(3-Chloro-4-fluorophenyl)-2-(cyclopropylmethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using cyclopropylmethanesulfonyl chloride. LC-MS: 384 (M+H)⁺.

Compound 37:N-(3-Chloro-4-fluorophenyl)-2-((3-methyloxetan-3-yl)methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using (3-methyloxetan-3-yl)methanesulfonyl chloride. LC-MS: 414 (M+H)⁺.

Compound 38:N-(3-Chloro-4-fluorophenyl)-2-(tetrahydro-2H-pyran-4-sulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using tetrahydro-2H-pyran-4-sulfonyl chloride. LC-MS: 414 (M+H)⁺.

Compound 39:(±)-N-(3-Chloro-4-fluorophenyl)-2-(tetrahydrofuran-3-sulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using tetrahydrofuran-3-sulfonyl chloride. LC-MS: 400 (M+H)⁺.

Compound 40:(±)-N-(3-Chloro-4-fluorophenyl)-2-(3-hydroxy-2-methylpropylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using methyl 3-(chlorosulfonyl)-2-methylpropanoate followed by LiBH₄reduction. LC-MS: 402 (M+H)⁺.

Compound 41:N-(3-Chloro-4-fluorophenyl)-2-(1-methylethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 2using propane-2-sulfonamide as a starting material. LC-MS: 372 (M+H)⁺.

Compound 42:N-(3-Chloro-4-fluorophenyl)-2-(2,2-dimethylpropylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 2using 2,2-dimethylpropane-1-sulfonamide as a starting material. LC-MS:400 (M+H)⁺.

Compound 43:N-(3,4-Difluorophenyl)-2-(1-methylethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 2using propane-2-sulfonamide and Intermediate C as starting materials.LC-MS: 356 (M+H)⁺.

Compound 44:N-(3,4-Difluorophenyl)-2-(tetrahydro-2H-pyran-4-sulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using tetrahydro-2H-pyran-4-sulfonyl chloride and Intermediate G. LC-MS:398 (M+H)⁺.

Compound 45:(±)-N-(3,4-Difluorophenyl)-2-(tetrahydrofuran-3-sulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using tetrahydrofuran-3-sulfonyl chloride and Intermediate G. LC-MS: 384(M+H)⁺

Compound 46:(±)-N-(3-Chloro-4-fluorophenyl)-2-((tetrahydrofuran-2-yl)methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using (tetrahydrofuran-2-yl)methanesulfonyl chloride. LC-MS: 414 (M+H)⁺.

Compound 47:(±)-N-(3-Chloro-4-fluorophenyl)-2-((tetrahydro-2H-pyran-2-yl)methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using (tetrahydro-2H-pyran-2-yl)methanesulfonyl chloride. LC-MS: 428(M+H)⁺.

Compound 48:(±)-N-(3-Chloro-4-fluorophenyl)-2-((tetrahydro-2H-pyran-3-yl)methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 2using (tetrahydro-2H-pyran-3-yl)methanesulfonamide. LC-MS: 428 (M+H)⁺.

Compound 49:(cis/trans)-N-(3-Chloro-4-fluorophenyl)-2-(4-hydroxycyclohexanesulfonamido)isonicotinamide

Step 1;

To a RBF in a 0° C. ice bath was added sodium chlorite (1.03 g, 11.34mmol) and anhydrous acetonitrile (10 mL) followed by concentrated HCl(2.27 mL) dropwise. To the cold stirring mixture was added4-mercaptocyclohexanol (0.50 g, 3.78 mmol, dissolved in 3 mL ofacetonitrile) dropwise. The ice bath was removed and the resultingmixture was stirred at RT for 2 h then diluted with EtOAc and water. Theorganics were washed sequentially with water, brine, dried over Na₂SO₄,filtered and concentrated in vacuo. The resulting crude oil containing4-hydroxycyclohexane-1-sulfonyl chloride was used as is in the nextstep.

Step 2;

4-hydroxycyclohexane-1-sulfonyl chloride (0.70 g, crude) was dissolvedin DCM (15 mL) and stirred at −78° C. To the cold stirring mixture wascondensed liquid ammonia (15 mL) and the resulting mixture was stirredat −78° C. for 2 h, refluxed at 0° C. for 2 h, then allowed to warm tort for 16 h. The solvents were evaporated in vacuo and the resultingcrude solid containing 4-hydroxycyclohexane-1-sulfonamide was used as isin the next step.

Step 3;

The title compound was prepared in an analogous manner to Compound 2using 4-hydroxycyclohexane-1-sulfonamide and CuI coupling conditions.LC-MS: 428 (M+H)⁺.

Compound 50:N-(3-Chloro-4-fluorophenyl)-2-(2-(pyridin-2-yl)ethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using 2-(pyridin-2-yl)ethanesulfonyl chloride hydrochloride as startingmaterial. LC-MS: 428 (M+H)⁺.

Compound 51:N-(3-Chloro-4-fluorophenyl)-2-(2-(4-methoxyphenyl)ethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using 2-(4-methoxyphenyl)ethanesulfonyl chloride as starting material.LC-MS: 464 (M+H)⁺.

Compound 52:N-(3,4-Difluorophenyl)-2-(2-(pyridin-2-yl)ethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using 2-(pyridin-2-yl)ethanesulfonyl chloride hydrochloride andIntermediate E as starting materials. LC-MS: 419 (M+H)⁺.

Compound 53:N-(3,4-Difluorophenyl)-2-(2-(4-methoxyphenyl)ethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using 2-(4-methoxyphenyl)ethanesulfonyl chloride and Intermediate E asstarting materials. LC-MS: 448 (M+H)⁺.

Compound 54:N-(3-Chloro-4-fluorophenyl)-2-((4-cyanophenyl)methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using (4-cyanophenyl)methanesulfonyl chloride as starting material.LC-MS: 445 (M+H)⁺.

Compound 55:2-((4-Cyanophenyl)methylsulfonamido)-N-(3,4-difluorophenyl)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using (4-cyanophenyl)methanesulfonyl chloride and Intermediate E asstarting materials. LC-MS: 429 (M+H)⁺.

Compound 56:N-(3-Chloro-4-fluorophenyl)-2-((tetrahydro-2H-pyran-4-yl)methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 2using (tetrahydro-2H-pyran-4-yl)methanesulfonyamide. LC-MS: 428 (M+H)⁺.

Compound 57:(±)-N-(3-Chloro-4-fluorophenyl)-2-(tetrahydro-2H-pyran-3-sulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 2using tetrahydro-2H-pyran-3-sulfonamide. LC-MS: 414 (M+H)⁺.

Compound 58:N-(3-Chloro-4-fluorophenyl)-2-((4-ethylphenyl)methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 2using (4-ethylphenyl)methanesulfonamide. LC-MS: 448 (M+H)⁺.

Compound 59:N-(3,4-Difluorophenyl)-2-((4-ethylphenyl)methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 2using (4-ethylphenyl)methanesulfonamide and Intermediate C. LC-MS: 432(M+H)⁺.

Compound 60:(±)-N-(3-Chloro-4-fluorophenyl)-2-(3-hydroxycyclohexanesulfonamido)isonicotinamide

Step 1;

To a solution of cyclohex-2-enone (2.00 g, 20.81 mmol) in DCE (100 mL)at RT was added indium chloride (0.23 g, 1.04 mmol) and thioacetic acid(2.38 mL, 31.21 mmol). The resulting mixture was stirred at 60° C. for16 h then diluted with EtOAc and brine. The organics were washed withwater, brine, dried over Na₂SO₄, filtered, and concentrated in vacuo.The resulting crude material was purified on silica gel eluting with asolvent gradient of 0% to 40% EtOAc in hexanes to afford pureS-(3-oxocyclohexyl)ethanethioate as an oil.

Step 2;

To a solution of S-(3-oxocyclohexyl)ethanethioate (1.00 g, 5.81 mmol) inMeOH (50 mL) at 0° C. was added NaBH₄ (0.27 g, 6.97 mmol) The resultingmixture was stirred at 0° C. for 1.5 h then diluted with EtOAc and waterafter reducing the volume of MeOH with a stream of nitrogen gas. Theorganics were washed with water twice and concentrated in vacuo. Theresulting crude material containing S-(3-hydroxycyclohexyl)ethanethioate was carried on as is into the next step.

Step 3;

Combined S-(3-hydroxycyclohexyl)ethanethioate (0.36 g, 2.07 mmol), THF(7.5 mL), MeOH (7.5 mL), water (4.2 mL), and 5 M NaOH (3.31 mL, 16.53mmol) and stirred at RT for 48 h. The reaction was quenched with a 20%aq. solution of citric acid monohydrate, the organics were partiallyevaporated, and remaining solution was diluted with EtOAc and brine. Theorganics were washed twice with water and concentrated in vacuo. Theresulting crude was coevaporated with EtOAc to afford3-mercaptocyclohexanol as an oil and was used as is in the next step.

Step 4;

To a RBF in a 0° C. ice bath was added sodium chlorite (0.67 g, 7.37mmol) and anhydrous acetonitrile (10 mL) followed by concentrated HCl(1.50 mL) dropwise. To the cold stirring mixture was added3-mercaptocyclohexanol (0.33 g, 2.46 mmol, dissolved in 3 mL ofacetonitrile) dropwise. The ice bath was removed and the resultingmixture was stirred at RT for 2 h then diluted with EtOAc and water. Theorganics were washed sequentially with water, brine, dried over Na₂SO₄,filtered and concentrated in vacuo. The resulting scentless crude oilcontaining 3-hydroxycyclohexane-1-sulfonyl chloride was used as is inthe next step.

Step 5;

3-Hydroxycyclohexane-1-sulfonyl chloride (˜0.40 g, crude) was dissolvedin DCM (10 mL) and stirred at −78° C. To the cold stirring mixture wascondensed liquid ammonia (10 mL) and the resulting mixture was stirredat −78° C. for 2 h, refluxed at 0° C. for 2 h, then allowed to warm tort for 16 h. The solvents were evaporated in vacuo, coevaporated with1:1 MeOH/EtOAc, and the resulting crude solid containing3-hydroxycyclohexane-1-sulfonamide was used as is in the next step.

Step 6;

The title compound was prepared in an analogous manner to Compound 2using 3-hydroxycyclohexane-1-sulfonamide and CuI coupling conditions.LC-MS: 428 (M+H)⁺.

Compound 61:(±)-N-(3-Chloro-4-fluorophenyl)-2-(3-hydroxycycloheptanesulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 61using cyclohept-2-enone in step 1. LC-MS: 442 (M+H)⁺.

Compound 62:2-(1-Benzylcyclopropanesulfonamido)-N-(3-chloro-4-fluorophenyl)isonicotinamide

Step 1:

To a solution of tert-butylamine (29.6 mL, 0.282 mol) in anhydrous THF(250 mL), cooled to −20° C. was added 3-chloropropylsulfonylchloride (25g, 0.141 mol) over a period of 10 minutes. The reaction mixture wasstirred vigorously at room temperature for 16 hrs after which it wasfiltered under vacuum. Concentration of the filtrate afforded acolorless oil which was dissolved in DCM, washed sequentially with 1NHCl then water, the organic phase dried (MgSO₄), filtered and evaporatedin vacuo. The resulting solid was triturated from a DCM/Hexanes mixtureto afford N-(tert-butyl)-3-chloropropane-1-sulfonamide as a colorlesssolid after filtration and drying in vacuo.

Step 2:

To a solution of N-(tert-butyl)-3-chloropropane-1-sulfonamide (2.5 g,11.7 mmol) in anhydrous THF (100 mL) cooled to −78° C. was addedn-butyllithium (2.5M in hexanes; 10 mL, 25.0 mmol). The resultingsolution was allowed to warm to room temperature, stirred for 1.5 hrsthen re-cooled to −78° C. after which a further addition ofn-butyllithium (2.5M in hexanes; 5 mL, 12.5 mmol) was made. Afterfurther warming to room temperature, the solution was re-cooled to −78°C. and benzyl bromide (1.5 mL, 12.6 mmol) added. The final solution wasstirred at room temperature for 12 hrs after which sat′d. aq. NH₄Clsolution was added and the resulting mixture extracted with EtOAc (2×),the organic phases dried (MgSO₄), filtered and evaporated in vacuo.Trituration of the residue with hexanes and filtration afforded1-benzyl-N-(tert-butyl)cyclopropane-1-sulfonamide as a colorless solid.

Step 3:

To 1-benzyl-N-(tert-butyl)cyclopropane-1-sulfonamide (2.0 g, 7.5 mmol)was added trifluoroacetic acid (30 mL). After stirring at roomtemperature for 16 hrs, the reaction mixture was evaporated to drynessand the resulting solid triturated from a minimum of EtOAc/hexanemixture to afford 1-benzylcyclopropane-1-sulfonamide as a colorlesssolid.

Step 4:

The title compound was prepared in an analogous manner to Compound 2using the prepared 1-benzylcyclopropane-1-sulfonamide. LC-MS: 460(M+H)⁺.

Compound 63:2-(1-Benzylcyclopropanesulfonamido)-N-(3,4-difluorophenyl)isonicotinamide

The title compound was prepared in an analogous manner to Compound 2using the prepared 1-benzylcyclopropane-1-sulfonamide and IntermediateC. LC-MS: 444 (M+H)⁺.

Compound 64:(±)-N-(3-Chloro-4-fluorophenyl)-2-(3-hydroxycyclopentanesulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 61using cyclopent-2-enone in step 1. LC-MS: 414 (M+H)⁺.

Compound 65:3-(N-(4-((3-Chloro-4-fluorophenyl)carbamoyl)pyridin-2-yl)sulfamoyl)benzoicacid

The title compound was prepared in an analogous manner to Compound 8using (4-cyanophenyl)methanesulfonyl chloride as starting material andthen standard ester hydrolysis conditions to afford the acid. LC-MS: 448(M−H)⁻.

Compound 66:N-(3-Chloro-4-fluorophenyl)-2-((4-isopropylphenyl)methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 2using (4-isopropylphenyl)methanesulfonamide. LC-MS: 462 (M+H)⁺.

Compound 67:N-(3,4-Difluorophenyl)-2-((4-isopropylphenyl)methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 2using (4-isopropylphenyl)methanesulfonamide and Intermediate C asstarting materials. LC-MS: 446 (M+H)⁺.

Compound 68:N-(3-Chloro-4-fluorophenyl)-2-(naphthalen-2-ylmethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using naphthalen-2-ylmethanesulfonyl chloride as starting material.LC-MS: 470 (M+H)⁺.

Compound 69:N-(3,4-Difluorophenyl)-2-(naphthalen-2-ylmethylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using naphthalen-2-ylmethanesulfonyl chloride and Intermediate E asstarting materials. LC-MS: 470 (M+H)⁺.

Compound 70:N-(3-Chloro-4-fluorophenyl)-2-((4-chlorophenyl)methylsulfonamido)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using (4-chlorophenyl)methanesulfonyl chloride as starting material.LC-MS: 454 (M+H)⁺.

Compound 71:2-((4-Chlorophenyl)methylsulfonamido)-N-(3,4-difluorophenyl)isonicotinamide

The title compound was prepared in an analogous manner to Compound 8using (4-chlorophenyl)methanesulfonyl chloride and Intermediate E asstarting materials. LC-MS: 438 (M+H)⁺.

Compound 72:2-((4-(tert-Butyl)phenyl)methylsulfonamido)-N-(3-chloro-4-fluorophenyl)isonicotinamide

Step 1:

To a solution of 1-(tert-butyl)-4-(chloromethyl)benzene (1.0 g, 5.47mmol) in DMSO (10 mL) was added sodium3-methoxy-3-oxopropane-1-sulfinate (see Baskin, J. M.; Wang, Z. Tet.Lett. 2002, 43, 8479; 1.14 g, 6.55 mmol) and stirred vigorously for 18hrs. NaOMe (25% wt; 1.5 mL) was added and after stirring for 30 min, thesolution was cooled in an ice bath and a premixed solution ofhydroxylamine-O-sulfonic acid (3.1 g, 27.4 mmol), NaOAc (1.7 g, 20.7mmol) in water (25 mL) was added. The resulting solution was stirred atroom temperature for 18 hrs after which it was extracted with EtOAc, theorganic phase separated, dried (MgSO₄), filtered and evaporated invacuo. The residue was then purified using silica gel chromatography toafford (4-(tert-butyl)phenyl)methanesulfonamide as a colorless solid.

Step 2:

The title compound was prepared in an analogous manner to Compound 2using (4-(tert-butyl)phenyl)methanesulfonamide as starting material.LC-MS: 476 (M+H)⁺.

Compound 73:2-((4-(tert-Butyl)phenyl)methylsulfonamido)-N-(3,4-difluorophenyl)isonicotinamide

The title compound was prepared in an analogous manner to Compound 2using (4-(tert-butyl)phenyl)methanesulfonamide and Intermediate C asstarting materials. LC-MS: 460 (M+H)⁺.

Compound 74:N-(3-Chloro-4-fluorophenyl)-2-((4-(2-hydroxypropan-2-yl)phenyl)methylsulfonamido)isonicotinamide

Step 1:

2-(p-Tolyl)propan-2-ol (90% Tech; 3.0 g, 19.9 mmol) was dissolved inCCl₄ and N-bromosuccinimide (3.48 g, 19.6 mmol) and benzoylperoxide (100mg, cat) was added. The reaction mixture was heated to reflux for 4 hrs,cooled and filtered. The filtrate was evaporated and the residuepurified on silica gel eluting with 0 to 10% EtOAc in hexanes to afford2-(4-(bromomethyl)phenyl)propan-2-ol as a colorless oil.

Step 2:

(4-(2-hydroxypropan-2-yl)phenyl)methanesulfonamide is prepared in anidentical fashion to Step 1 as described for Compound 72.

Step 3:

The title compound was prepared in an analogous manner to Compound 2using (4-(2-hydroxypropan-2-yl)phenyl)methanesulfonamide as startingmaterial. LC-MS: 478 (M+H)⁺.

Compound 75:4-fluoro-N-(4-fluoro-3-methylphenyl)-3-(1-methyl-1H-pyrazole-3-sulfonamido)benzamide

Step 1:

To a solution of 3-amino-4-fluorobenzoic acid (2.0 g, 7.7 mmol) indichloromethane (20 mL) was added 4-fluoro-3-methylaniline (968 mg, 7.7mmol), diisopropylethylamine (2.68 mL, 15.4 mmol) and HATU (3.22 g, 8.47mmol). The reaction mixture was maintained at room temperature for 12hrs after which water was added and diluted with dichloromethane. Theresulting suspension was filtered and the solids washed withdichloromethane and dried in vacuo to afford 1.39 g of3-amino-4-fluoro-N-(4-fluoro-3-methylphenyl)benzamide as a colorlesssolid. Purification of the filtrate using column chromatography elutingwith 0 to 100% EtOAc in hexanes afforded a further 1.0 g of the desiredamide.

Step 2:

To a solution of 3-Amino-4-fluoro-N-(4-fluoro-3-methylphenyl)benzamide(110 mg, 0.42 mmol) in pyridine (3 mL) cooled to 0° C. was added1-methyl-1H-pyrazole-3-sulfonyl chloride (90 mg, 0.50 mmol) andcatalytic DMAP (10 mg). After stirring at room temperature for 16 hrs,the solution was diluted with EtOAc and water, the organic phaseseparated, washed with brine, dried (MgSO₄), filtered and evaporated invacuo. Purification of the residue using preparative HPLC afforded thetitle compound.

Compound 76:N-(3-chloro-4-fluorophenyl)-3-(2-methoxyethylsulfonamido)benzamide

Prepared in an identical manner to Compound 75, step 2 usingIntermediate I as starting material and 2-methoxyethanesulfonylchloride.

Compound 77:N-(3-chloro-4-fluorophenyl)-3-(thiophene-2-sulfonamido)benzamide

Prepared in an identical manner to Compound 75, step 2 usingIntermediate I as starting material and thiophene-2-sulfonyl chloride.

Compound 78:N-(3-chloro-4-fluorophenyl)-3-(1H-pyrazole-4-sulfonamido)benzamide

Prepared in an identical manner to Compound 75, step 2 usingIntermediate I as starting material and 1H-pyrazole-4-sulfonyl chloride.

Compound 79:N-(3-chloro-4-fluorophenyl)-3-(3,5-dimethyl-1H-pyrazole-4-sulfonamido)benzamide

Prepared in an identical manner to Compound 75, step 2 usingIntermediate I as starting material and3,5-dimethyl-1H-pyrazole-4-sulfonyl chloride.

Compound 80:(±)-N-(3-chloro-4-fluorophenyl)-3-(tetrahydrofuran-3-sulfonamido)benzamide

Prepared in an identical manner to Compound 75, step 2 usingIntermediate I as starting material and tetrahydrofuran-3-sulfonylchloride.

Compound 81:(±)-N-(3-chloro-4-fluorophenyl)-3-((tetrahydrofuran-2-yl)methylsulfonamido)benzamide

Prepared in an identical manner to Compound 75, step 2 usingIntermediate I as starting material and(tetrahydrofuran-2-yl)methanesulfonyl chloride.

Compound 82:(±)-N-(3-chloro-4-fluorophenyl)-3-(2-methoxy-1-methylethylsulfonamido)benzamide

Prepared in an identical manner to Compound 75, step 2 usingIntermediate I as starting material and 1-methoxypropane-2-sulfonylchloride.

Compound 83:(±)-N-(3-chloro-4-fluorophenyl)-3-(2-methyltetrahydrofuran-3-sulfonamido)benzamide

Prepared in an identical manner to Compound 75, step 2 usingIntermediate I as starting material and2-methyltetrahydrofuran-3-sulfonyl chloride.

Compound:(±)-N-(3-chloro-4-fluorophenyl)-3-(4-hydroxy-1,1-dioxidotetrahydrothiophene-3-sulfonamido)benzamide

Prepared in an identical manner to Compound 75, step 2 usingIntermediate I as starting material and4-hydroxytetrahydrothiophene-3-sulfonyl chloride 1,1-dioxide.

Compound 85:3-(4-(benzyloxy)piperidine-1-sulfonamido)-N-(3-chloro-4-fluorophenyl)benzamide

Step 1:

Preparation of 4-(benzyloxy)piperidine-1-sulfonyl chloride; To an icecooled solution of 4-(benzyloxy)piperidine hydrochloride (0.5 g, 2.2mmol) in DCM (10 mL) was added triethylamine (920 μL, 6.6 mmol) followedby dropwsie addition of chlorosulfonic acid (146 μL, 2.2 mmol). Theresulting solution was stirred at room temperature for 16 hrs andconcentrated, the residue washed with Et₂O and dried in vacuo. The cruderesidue was suspended in benzene (7 mL), PCl₅ (458 mg, 2.2 mmol) addedand heated to reflux for 2 hrs. After cooling, the mixture was dilutedin EtOAc and washed with 5% citric acid solution, saturated bicarbonate,and brine. After drying with MgSO₄, the crude material was concentratedto afford 4-(benzyloxy)piperidine-1-sulfonyl chloride as an oil usedwithout further purification.

Step 2:

Prepared in an identical manner to Compound 75, step 2 usingIntermediate I as starting material and4-(benzyloxy)piperidine-1-sulfonyl chloride.

The compounds of the invention may possess one or more stereocenters,and each stereocenter may exist independently in either the R or Sconfiguration. In one embodiment, compounds described herein are presentin optically active or racemic forms. It is to be understood that thecompounds described herein encompass racemic, optically-active,regioisomeric and stereoisomeric forms, or combinations thereof thatpossess the therapeutically useful properties described herein.

Preparation of optically active forms is achieved in any suitablemanner, including by way of non-limiting example, by resolution of theracemic form with recrystallization techniques, synthesis fromoptically-active starting materials, chiral synthesis, orchromatographic separation using a chiral stationary phase. In oneembodiment, a mixture of one or more isomer is utilized as thetherapeutic compound described herein. In another embodiment, compoundsdescribed herein contain one or more chiral centers. These compounds areprepared by any means, including stereoselective synthesis,enantioselective synthesis and/or separation of a mixture of enantiomersand/or diastereomers. Resolution of compounds and isomers thereof isachieved by any means including, by way of non-limiting example,chemical processes, enzymatic processes, fractional crystallization,distillation, and chromatography.

The methods and formulations described herein include the use ofN-oxides (if appropriate), crystalline forms (also known as polymorphs),solvates, amorphous phases, and/or pharmaceutically acceptable salts ofcompounds having the structure of any compound of the invention, as wellas metabolites and active metabolites of these compounds having the sametype of activity. Solvates include water, ether (e.g., tethrahydrofuran,methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetatesand the like. In one embodiment, the compounds described herein exist insolvated forms with pharmaceutically acceptable solvents such as water,and ethanol. In another embodiment, the compounds described herein existin unsolvated form.

In one embodiment, the compounds of the invention may exist astautomers. All tautomers are included within the scope of the compoundspresented herein.

Materials:

Unless otherwise noted, all starting materials and resins were obtainedfrom commercial suppliers and used without purification.

Example: Inhibition of HBV Replication Dot-Blot Assay

Compounds active in the HBV assembly assay are tested for their activityand toxicity in cellular assay. In the first anti-viral assay, theability of compounds to inhibit HBV replication in an HBV-producinghepatoma cell line using the dot-blot method is evaluated.

Briefly, confluent monolayers of HepG2-2.2.15 cells are incubated withcomplete medium containing various concentrations of a test compound.Three days later, the culture medium is replaced with fresh mediumcontaining the appropriately diluted test compound. Six days followingthe initial administration of the test compound, the cell culturesupernatant is collected, and cell lysis is performed. The samples areapplied onto Nylos membranes and DNA is immobilized to the membrane byUV cross-linking. After pre-hybridization, the HBV probe is added andthe hybridization is performed overnight. The membranes are exposed tothe KODAK films; antiviral activity is calculated from the reduction inHBV DNA levels (EC₅₀). The EC₅₀ for antiviral activity is calculatedfrom the dose response curves of active compounds. Assay performanceover time is monitored by the use of the standard positive controlcompounds ETV, BAY 41-4109, and HAP-1.

Compound cytotoxity (TC₅₀) is measured in this same HepG2-2.2.15 cellline using a CELLTITER BLUE-based cytotoxicity assay employed asrecommended by manufacturer (Promega). To confirm and expand theseresults, a second antiviral assay is carried out on active compoundsusing the stable HBV cell line HepG2.2.15 and measuring anti-HBV potencyby real-time PCR and cytotoxicity by CELLTITER BLUE. In this assay, 24hours after cell seeding, HepG2-2.2.15 cells are incubated with completemedium containing various concentrations of a test compound with BAY41-4109 and HAP-1 used as positive controls. After three days, theculture medium is replaced with fresh medium containing theappropriately diluted test compound. The cell culture is collected sixdays following the initial administration of the test compound, followedby HBV DNA extraction using QIAamp 96 DNA Blood Kit (Qiagen). Theextracted HBV DNA is diluted and analyzed by Real-Time PCR. A standardcurve is generated by plotting Ct value vs the amount of HBV plasmidstandard. Cytotoxicity is determined similarly to the above describedmethod by applying a dye uptake method (CELLTITER BLUE kit, Promega).

Selected compounds are tested for their activity and toxicity incellular assay. In the first anti-viral assay, the ability of compoundsto inhibit HBV replication in an HBV-producing hepatoma cell line usingthe dot-blot method is evaluated.

Confluent monolayers of HepG2-2.2.15 cells are incubated with completemedium containing various concentrations of a test compound. Three dayslater, the culture medium is replaced with fresh medium containing theappropriately diluted test compound. Six days following the initialadministration of the test compound, the cell culture supernatant iscollected, and cell lysis was performed. The samples are applied ontoNylos membranes and DNA is immobilized to the membrane by UVcross-linking. After pre-hybridization, the HBV probe is added and thehybridization is performed overnight. The membranes are exposed to theKODAK films; antiviral activity is calculated from the reduction in HBVDNA levels (EC₅₀). The EC₅₀ for antiviral activity is calculated fromthe dose response curves of active compounds. Assay performance overtime is monitored by the use of the standard positive control compoundsETV, BAY 41-4109, and HAP-1. Results for selected compounds of theinvention are illustrated in Table 2.

Cytotoxity (CC₅₀) is measured in this same HepG2-2.2.15 cell line usinga CELLTITER BLUE-based cytotoxicity assay employed as recommended by themanufacturer (Promega).

Example: HBV Replication Inhibition Assay

HBV replication inhibition by the compounds of this invention could bedetermined in cells infected or transfected with HBV, or cells withstably integrated HBV, such as HepG2.2.15 cells (Sells et al. 1987). Inthis example, HepG2.2.15 cells are maintained in cell culture mediumcontaining 10% fetal bovine serum (FBS), Geneticin, L-glutamine,penicillin and streptomycin. HepG2.2.15 cells could be seeded in 96-wellplates at a density of 40,000 cells/well and be treated with seriallydiluted compounds at a final DMSO concentration of 0.5% either alone orin combination by adding drugs in a checker box format. Cells areincubated with compounds for three days, after which medium is removedand fresh medium containing compounds is added to cells and incubatedfor another three days. At day 6, supernatant is removed and treatedwith DNase at 37° C. for 60 minutes, followed by enzyme inactivation at75° C. for 15 minutes. Encapsidated HBV DNA is released from the virionsand covalently linked HBV polymerase by incubating in lysis buffer(Affymetrix QS0010) containing 2.5 μg proteinase K at 50° C. for 40minutes. HBV DNA is denatured by addition of 0.2 M NaOH and detectedusing a branched DNA (bDNA) QuantiGene assay kit according tomanufacturer recommendation (Affymetrix).

HBV DNA levels could also be quantified using qPCR, based onamplification of encapsidated HBV DNA extraction with QUICKEXTRACTSolution (Epicentre Biotechnologies) and amplification of HBV DNA usingHBV specific PCR probes that can hybridize to HBV DNA and afluorescently labeled probe for quantitation. In addition, cellviability of HepG2.2.15 cells incubated with test compounds alone or incombination is determined by using CELLTITER-GLO reagent according tothe manufacturer protocol (Promega). The mean background signal fromwells containing only culture medium is subtracted from all othersamples, and percent inhibition at each compound concentration iscalculated by normalizing to signals from HepG2.2.15 cells treated with0.5% DMSO using equation E1.

% inhibition=(DMSOave−Xi)/DMSOave×100%  E1:

wherein DMSOave is the mean signal calculated from the wells that aretreated with DMSO control (0% inhibition control) and Xi is the signalmeasured from the individual wells. EC50 values, effectiveconcentrations that achieved 50% inhibitory effect, are determined bynon-linear fitting using Graphpad Prism software (San Diego, Calif.) andequation E2.

Y=Ymin+(Ymax−Ymin)/(1+10(Log EC50−X)×HillSlope)  E2:

-   -   wherein Y represents percent inhibition values and X represents        the logarithm of compound concentrations.

Selected compounds of the invention were assayed in the HBV replicationassay, as described above and a representative group of these activecompounds is shown in Table 3.

TABLE 2 “Activity” represents activity in Dot-Blot assay (‘+’ indicatesEC₅₀ <10 μM) Compound No. Activity 1 + 2 + 3 + 5 + 6 + 8 + 9 + 11 + 12 +15 + 16 + 17 + 19 + 21 + 22 + 23 + 27 + 28 + 30 + 32 + 35 + 36 + 39 +41 + 42 + 43 + 51 + 57 + 58 + 59 + 64 + 75 + 76 + 77 + 78 + 79 + 80 +81 + 82 + 83 + 84 + 85 +

TABLE 3 HBV Replication Inhibition Compound Extra HBV DNA No (μM) 1 3 20.1 3 2.1 4 1 58 0.3 59 0.7 64 2.7

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1-2. (canceled)
 3. A compound of Formula II:

or a pharmaceutically acceptable salt thereof; wherein each R¹ isindependently selected from H, halo, and C₁₋₆-alkyl; R² is halo; R³ ishalo; R⁴ is C₁₋₆-heteroalkyl or heteroaryl, each of which may beindependently substituted with one or two of C₁₋₆-alkyl; R⁵ is H; and nis 0 or
 1. 4. The compound of claim 3, or a pharmaceutically acceptablesalt thereof, wherein the compound is selected from the group consistingof:

5-10. (canceled)
 11. A composition comprising a compound according toclaim 3, or a salt, solvate, or N-oxide thereof, further comprising atleast one pharmaceutically acceptable carrier.
 12. A method of treatingan HBV infection in an individual in need thereof, comprisingadministering to the individual a therapeutically effective amount of acompound according to claim
 3. 13. The method of claim 12, furthercomprising administering to the individual at least one additionaltherapeutic agent selected from the group consisting of an HBV vaccine,HBV polymerase inhibitor, interferon, pegylated interferon, viral entryinhibitor, viral maturation inhibitor, BAY 41-4109, reversetranscriptase inhibitor, a TLR-agonist, AT-61((E)-N-(1-chloro-3-oxo-1-phenyl-3-(piperidin-1-yl)prop-1-en-2-yl)benzamide),and AT-130((E)-N-(1-bromo-1-(2-methoxyphenyl)-3-oxo-3-(piperidin-1-yl)prop-1-en-2-yl)-4-nitrobenzamide),and a combination thereof.
 14. The method of claim 13, wherein thepegylated interferon is pegylated interferon alpha (IFN-α), pegylatedinterferon lambda (IFN-λ), or pegylated interferon gamma (IFN-γ). 15.The method of claim 13, wherein the reverse transcriptase inhibitor isat least one of Zidovudine, Didanosine, Zalcitabine, ddA, Stavudine,Lamivudine, Abacavir, Emtricitabine, Entecavir, Apricitabine,Atevirapine, ribavirin, acyclovir, famciclovir, valacyclovir,ganciclovir, valganciclovir, Tenofovir, Adefovir, cidofovir, Efavirenz,Nevirapine, Delavirdine, or Etravirine.
 16. The method of claim 13,wherein the TLR-agonist is selected from the group consisting ofSM360320 (9-benzyl-8-hydroxy-2-(2-methoxy-ethoxy)adenine) and AZD 8848(methyl[3-({[3-(6-amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-9-yl)propyl][3-(4-morpholinyl)propyl]amino}methyl)phenyl]acetate).